Nucleic Acid Molecules and Other Molecules Associated with Plants

ABSTRACT

Expressed Sequence Tags (ESTs) isolated from soybean are disclosed. The ESTs provide a unique molecular tool for the targeting and isolation of novel genes for plant protection and improvement. The disclosed ESTs have utility in the development of new strategies for understanding critical plant developmental and metabolic pathways. The disclosed ESTs have particular utility in isolating genes and promoters, identifying and mapping the genes involved in developmental and metabolic pathways, and determining gene function. Sequence homology analyses using the ESTs provided in the present invention, will result in more efficient gene screening for desirable agronomic traits. An expanding database of these select pieces of the plant genomics puzzle will quickly expand the knowledge necessary for subsequent functional validation, a key limitation in current plant biotechnology efforts.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority under 35 U.S.C. § 119(e) toprovisional application 60/067,000, filed Nov. 24, 1997, the entirety ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is in the field of plant biochemistry. Morespecifically the invention relates to nucleic acid molecules that encodeproteins and fragments of proteins produced in plant cells, inparticular, soybean plants. The invention also relates to proteins andfragments of proteins so encoded and antibodies capable of binding theproteins. The invention also relates to methods of using the nucleicacid molecules, proteins and fragments of proteins.

BACKGROUND OF THE INVENTION I. Expressed Sequence Tag Nucleic AcidMolecules

Expressed sequence tags, or ESTs, are short sequences of randomlyselected clones from a cDNA (or complementary DNA) library which arerepresentative of the cDNA inserts of these randomly selected clones.McCombie, et al., Nature Genetics, 1:124-130 (1992); Kurata, et al.,Nature Genetics, 8: 365-372 (1994); Okubo, et al., Nature Genetics, 2:173-179 (1992), all of which references are incorporated herein in theirentirety.

Using conventional methodologies, cDNA libraries can be constructed fromthe mRNA (messenger RNA) of a given tissue or organism using poly dTprimers and reverse transcriptase (Efstratiadis, et al., Cell 7:279-288(1976), the entirety of which is herein incorporated by reference;Higuchi, et al., Proc. Natl. Acad. Sci. (U.S.A.) 73:3146-3150 (1976),the entirety of which is herein incorporated by reference; Maniatis, etal., Cell 8:163 (1976) the entirety of which is herein incorporated byreference; Land, et al., Nucleic Acids Res. 9:2251-2266 (1981), theentirety of which is herein incorporated by reference; Okayama, et al.,Mol. Cell. Biol. 2:161-170 (1982), the entirety of which is hereinincorporated by reference; Gübler, et al., Gene 25:263 (1983), theentirety of which is herein incorporated by reference).

Several methods may be employed to obtain full-length cDNA constructs.For example, terminal transferase can be used to add homopolymeric tailsof dC residues to the free 3′ hydroxyl groups (Land, et al., NucleicAcids Res. 9:2251-2266 (1981), the entirety of which is hereinincorporated by reference). This tail can then be hybridized by a polydG oligo which can act as a primer for the synthesis of full lengthsecond strand cDNA. Okayama and Berg, report a method for obtaining fulllength cDNA constructs. This method has been simplified by usingsynthetic primer-adapters that have both homopolymeric tails for primingthe synthesis of the first and second strands and restriction sites forcloning into plasmids (Coleclough, et al., Gene 34:305-314 (1985), theentirety of which is herein incorporated by reference) and bacteriophagevectors (Krawinkel, et al., Nucleic Acids Res. 14:1913 (1986), theentirety of which is herein incorporated by reference; and Han, et al.,Nucleic Acids Res. 15:6304 (1987), the entirety of which is hereinincorporated by reference).

These strategies have been coupled with additional strategies forisolating rare mRNA populations. For example, a typical mammalian cellcontains between 10,000 and 30,000 different mRNA sequences. Davidson,Gene Activity in Early Development, 2nd ed., Academic Press, New York(1976). The number of clones required to achieve a given probabilitythat a low-abundance mRNA will be present in a cDNA library isN=(ln(1−P))/(ln(1−1/n)) where N is the number of clones required, P isthe probability desired, and 1/n is the fractional proportion of thetotal mRNA that is represented by a single rare mRNA. (Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press (1989), the entirety of which is herein incorporated byreference.).

A method to enrich preparations of mRNA for sequences of interest is tofractionate by size. One such method is to fractionate byelectrophoresis through an agarose gel (Pennica, et al., Nature301:214-221 (1983), the entirety of which is herein incorporated byreference). Another such method employs sucrose gradient centrifugationin the presence of an agent, such as methylmercuric hydroxide, thatdenatures secondary structure in RNA (Schweinfest, et al., Proc. Natl.Acad. Sci. (U.S.A.) 79:4997-5000 (1982), the entirety of which is hereinincorporated by reference).

A frequently adopted method is to construct equalized or normalized cDNAlibraries (Ko, Nucleic Acids Res. 18:5705-5711 (1990), the entirety ofwhich is herein incorporated by reference; Patanjali, S. R. et al.,Proc. Natl. Acad. Sci. (U.S.A.) 88:1943-1947 (1991), the entirety ofwhich is herein incorporated by reference). Typically, the cDNApopulation is normalized by subtractive hybridization. Schmid, et al.,J. Neurochem. 48:307-312 (1987) the entirety of which is hereinincorporated by reference; Fargnoli, et al., Anal. Biochem. 187:364-373(1990) the entirety of which is herein incorporated by reference;Travis, et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:1696-1700 (1988) theentirety of which is herein incorporated by reference; Kato, Eur. J.Neurosci. 2:704 (1990); and Schweinfest, et al., Genet. Anal. Tech.Appl. 7:64 (1990), the entirety of which is herein incorporated byreference). Subtraction represents another method for reducing thepopulation of certain sequences in the cDNA library. Swaroop, et al.,Nucleic Acids Res. 19:1954 (1991), the entirety of which is hereinincorporated by reference).

ESTs can be sequenced by a number of methods. Two basic methods may beused for DNA sequencing, the chain termination method of Sanger et al.,Proc. Natl. Acad. Sci. (U.S.A.) 74: 5463-5467 (1977), the entirety ofwhich is herein incorporated by reference and the chemical degradationmethod of Maxam and Gilbert, Proc. Nat. Acad. Sci. (U.S.A.) 74: 560-564(1977), the entirety of which is herein incorporated by reference.Automation and advances in technology such as the replacement ofradioisotopes with fluorescence-based sequencing have reduced the effortrequired to sequence DNA (Craxton, Methods, 2: 20-26 (1991), theentirety of which is herein incorporated by reference; Ju et al., Proc.Natl. Acad. Sci. (U.S.A.) 92: 4347-4351 (1995), the entirety of which isherein incorporated by reference; Tabor and Richardson, Proc. Natl.Acad. Sci. (U.S.A.) 92: 6339-6343 (1995), the entirety of which isherein incorporated by reference). Automated sequencers are availablefrom, for example, Pharmacia Biotech, Inc., Piscataway, N.J. (PharmaciaALF), LI-COR, Inc., Lincoln, Nebr. (LI-COR 4,000) and Millipore,Bedford, Mass. (Millipore BaseStation).

In addition, advances in capillary gel electrophoresis have also reducedthe effort required to sequence DNA and such advances provide a rapidhigh resolution approach for sequencing DNA samples (Swerdlow andGesteland, Nucleic Acids Res. 18:1415-1419 (1990); Smith, Nature349:812-813 (1991); Luckey et al., Methods Enzymol. 218:154-172 (1993);Lu et al., J. Chromatog. A. 680:497-501 (1994); Carson et al., Anal.Chem. 65:3219-3226 (1993); Huang et al., Anal. Chem. 64:2149-2154(1992); Kheterpal et al., Electrophoresis 17:1852-1859 (1996); Quesadaand Zhang, Electrophoresis 17:1841-1851 (1996); Baba, Yakugaku Zasshi117:265-281 (1997), all of which are herein incorporated by reference intheir entirety).

ESTs longer than 150 bases have been found to be useful for similaritysearches and mapping. (Adams, et al., Science 252:1651-1656 (1991),herein incorporated by reference.) EST sequences normally range from150-450 bases. This is the length of sequence information that isroutinely and reliably generated using single run sequence data.Typically, only single run sequence data is obtained from the cDNAlibrary, Adams, et al., Science 252:1651-1656 (1991). Automated singlerun sequencing typically results in an approximately 2-3% error or baseambiguity rate. (Boguski, et al., Nature Genetics, 4:332-333 (1993), theentirety of which is herein incorporated by reference).

EST databases have been constructed or partially constructed from, forexample, C. elegans (McCombrie, et al., Nature Genetics 1:124-131(1992), human liver cell line HepG2 (Okubo, et al., Nature Genetics2:173-179 (1992)), human brain RNA (Adams, et al., Science 252:1651-1656(1991); Adams, et al., Nature 355:632-635 (1992)), Arabidopsis, (Newman,et al., Plant Physiol. 106:1241-1255 (1994)); and rice (Kurata, et al.,Nature Genetics 8:365-372 (1994).

II. Sequence Comparisons

A characteristic feature of a protein or DNA sequence is that it can becompared with other known protein or DNA sequences. Sequence comparisonscan be undertaken by determining the similarity of the test or querysequence with sequences in publicly available or propriety databases(“similarity analysis”) or by searching for certain motifs (“intrinsicsequence analysis”) (e.g. cis elements) (Coulson, Trends inBiotechnology, 12: 76-80 (1994), the entirety of which is hereinincorporated by reference; Birren, et al., Genome Analysis, 1: 543-559(1997), the entirety of which is herein incorporated by reference).

Similarity analysis includes database search and alignment. Examples ofpublic databases include the DNA Database of Japan (DDBJ)(http://www.ddbj.nig.ac.jp/); Genebank(http://www.ncbi.nlm.nih.gov/web/Genbank/Index.htlm); and the EuropeanMolecular Biology Laboratory Nucleic Acid Sequence Database (EMBL)(http://www.ebi.ac.uk/ebi_docs/embl_db.html). A number of differentsearch algorithms have been developed, one example of which are thesuite of programs referred to as BLAST programs. There are fiveimplementations of BLAST, three designed for nucleotide sequencesqueries (BLASTN, BLASTX, and TBLASTX) and two designed for proteinsequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology,12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).

BLASTN takes a nucleotide sequence (the query sequence) and its reversecomplement and searches them against a nucleotide sequence database.BLASTN was designed for speed, not maximum sensitivity, and may not finddistantly related coding sequences. BLASTX takes a nucleotide sequence,translates it in three forward reading frames and three reversecomplement reading frames, and then compares the six translationsagainst a protein sequence database. BLASTX is useful for sensitiveanalysis of preliminary (single-pass) sequence data and is tolerant ofsequencing errors (Gish and States, Nature Genetics, 3: 266-272 (1993),the entirety of which is herein incorporated by reference). BLASTN andBLASTX may be used in concert for analyzing EST data (Coulson, Trends inBiotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, 1:543-559 (1997).

Given a coding nucleotide sequence and the protein it encodes, it isoften preferable to use the protein as the query sequence to search adatabase because of the greatly increased sensitivity to detect moresubtle relationships. This is due to the larger alphabet of proteins (20amino acids) compared with the alphabet of nucleic acid sequences (4bases), where it is far easier to obtain a match by chance. In addition,with nucleotide alignments, only a match (positive score) or a mismatch(negative score) is obtained, but with proteins, the presence ofconservative amino acid substitutions can be taken into account. Here, amismatch may yield a positive score if the non-identical residue hasphysical/chemical properties similar to the one it replaced. Variousscoring matrices are used to supply the substitution scores of allpossible amino acid pairs. A general purpose scoring system is theBLOSUM62 matrix (Henikoff and Henikoff, Proteins, 17: 49-61 (1993), theentirety of which is herein incorporated by reference), which iscurrently the default choice for BLAST programs. BLOSUM62 is tailoredfor alignments of moderately diverged sequences and thus may not yieldthe best results under all conditions. Altschul, J. Mol. Biol. 36:290-300 (1993), the entirety of which is herein incorporated byreference, uses a combination of three matrices to cover allcontingencies. This may improve sensitivity, but at the expense ofslower searches. In practice, a single BLOSUM62 matrix is often used butothers (PAM40 and PAM250) may be attempted when additional analysis isnecessary. Low PAM matrices are directed at detecting very strong butlocalized sequence similarities, whereas high PAM matrices are directedat detecting long but weak alignments between very distantly relatedsequences.

Homologues in other organisms are available that can be used forcomparative sequence analysis. Multiple alignments are performed tostudy similarities and differences in a group of related sequences.CLUSTAL W is a multiple sequence alignment package available thatperforms progressive multiple sequence alignments based on the method ofFeng and Doolittle, J. Mol. Evol. 25: 351-360 (1987), the entirety ofwhich is herein incorporated by reference. Each pair of sequences isaligned and the distance between each pair is calculated; from thisdistance matrix, a guide tree is calculated, and all of the sequencesare progressively aligned based on this tree. A feature of the programis its sensitivity to the effect of gaps on the alignment; gap penaltiesare varied to encourage the insertion of gaps in probable loop regionsinstead of in the middle of structured regions. Users can specify gappenalties, choose between a number of scoring matrices, or supply theirown scoring matrix for both the pairwise alignments and the multiplealignments. CLUSTAL W for UNIX and VMS systems is available at:ftp.ebi.ac.uk. Another program is MACAW (Schuler et al., Proteins,Struct. Func. Genet, 9:180-190 (1991), the entirety of which is hereinincorporated by reference, for which both Macintosh and MicrosoftWindows versions are available. MACAW uses a graphical interface,provides a choice of several alignment algorithms, and is available byanonymous ftp at: ncbi.nlm.nih.gov (directory/pub/macaw).

Sequence motifs are derived from multiple alignments and can be used toexamine individual sequences or an entire database for subtle patterns.With motifs, it is sometimes possible to detect distant relationshipsthat may not be demonstrable based on comparisons of primary sequencesalone. Currently, the largest collection of sequence motifs in the worldis PROSITE (Bairoch and Bucher, Nucleic Acid Research, 22: 3583-3589(1994), the entirety of which is herein incorporated by reference.)PROSITE may be accessed via either the ExPASy server on the World WideWeb or anonymous ftp site. Many commercial sequence analysis packagesalso provide search programs that use PROSITE data.

A resource for searching protein motifs is the BLOCKS E-mail serverdeveloped by S. Henikoff, Trends Biochem Sci., 18:267-268 (1993), theentirety of which is herein incorporated by reference; Henikoff andHenikoff, Nucleic Acid Research, 19:6565-6572 (1991), the entirety ofwhich is herein incorporated by reference; Henikoff and Henikoff,Proteins, 17: 49-61 (1993). BLOCKS searches a protein or nucleotidesequence against a database of protein motifs or “blocks.” Blocks aredefined as short, ungapped multiple alignments that represent highlyconserved protein patterns. The blocks themselves are derived fromentries in PROSITE as well as other sources. Either a protein ornucleotide query can be submitted to the BLOCKS server; if a nucleotidesequence is submitted, the sequence is translated in all six readingframes and motifs are sought in these conceptual translations. Once thesearch is completed, the server will return a ranked list of significantmatches, along with an alignment of the query sequence to the matchedBLOCKS entries.

Conserved protein domains can be represented by two-dimensionalmatrices, which measure either the frequency or probability of theoccurrences of each amino acid residue and deletions or insertions ineach position of the domain. This type of model, when used to searchagainst protein databases, is sensitive and usually yields more accurateresults than simple motif searches. Two popular implementations of thisapproach are profile searches (such as GCG program ProfileSearch) andHidden Markov Models (HMMs) (Krough et al., J. Mol. Biol. 235:1501-1531(1994); Eddy, Current Opinion in Structural Biology 6:361-365 (1996),both of which are herein incorporated by reference in their entirety).In both cases, a large number of common protein domains have beenconverted into profiles, as present in the PROSITE library, or HHMmodels, as in the Pfam protein domain library (Sonnhammer et al.,Proteins 28:405-420 (1997), the entirety of which is herein incorporatedby reference). Pfam contains more than 500 HMM models for enzymes,transcription factors, signal transduction molecules, and structuralproteins. Protein databases can be queried with these profiles or HMMmodels, which will identify proteins containing the domain of interest.For example, HMMSW or HMMFS, two programs in a public domain packagecalled HMMER (Sonnhammer et al., Proteins 28:405-420 (1997)) can beused.

PROSITE and BLOCKS represent collected families of protein motifs. Thus,searching these databases entails submitting a single sequence todetermine whether or not that sequence is similar to the members of anestablished family. Programs working in the opposite direction compare acollection of sequences with individual entries in the proteindatabases. An example of such a program is the Motif Search Tool, orMoST (Tatusov et al. Proc. Natl. Acad. Sci. 91: 12091-12095 (1994), theentirety of which is herein incorporated by reference.) On the basis ofan aligned set of input sequences, a weight matrix is calculated byusing one of four methods (selected by the user); a weight matrix issimply a representation, position by position in an alignment, of howlikely a particular amino acid will appear. The calculated weight matrixis then used to search the databases. To increase sensitivity, newlyfound sequences are added to the original data set, the weight matrix isrecalculated, and the search is performed again. This procedurecontinues until no new sequences are found.

SUMMARY OF THE INVENTION

The present invention provides a substantially purified nucleic acidmolecule that encodes a soybean protein or fragment thereof comprising asequence selected from the group consisting of SEQ ID NO: 1 through SEQID NO: 5521.

The present invention also provides one or more substantially purifiednucleic acid molecule selected from the group consisting of a nucleicacid molecule comprising SEQ ID NO: 1 or complement thereof through SEQID NO:5521 or complement thereof.

The present invention also provides a substantially purified soybeanprotein or fragment thereof, wherein said soybean protein is encoded bya nucleic acid molecule that comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1 through SEQ ID NO: 5521.

The present invention further provides a substantially purified protein,peptide, or fragment thereof encoded by a nucleic acid sequence whichspecifically hybridizes to a nucleic acid molecule selected from thegroup consisting of SEQ ID NO: 1 through SEQ ID NO:5521.

The present invention further provides a substantially purified antibodycapable of specifically binding to a protein or fragment thereof encodedby a nucleic acid sequence which specifically hybridizes to a nucleicacid molecule selected from the group consisting of SEQ ID NO:1 throughSEQ ID NO:5521.

The present invention also provides a transformed plant transformed tocontain a nucleic acid molecule which comprises: (A) an exogeneouspromoter region which functions in plant cells to cause the productionof an mRNA molecule; which is linked to (B) a structural nucleic acidmolecule, wherein said structural nucleic acid molecule comprises anucleic acid molecule that encodes a protein, peptide, or fragmentthereof which hybridizes to SEQ ID NO:1 through SEQ ID NO:5521 expressedin an effective amount to produce a desirable agronomic effect; which islinked to (C) a 3′ non-translated sequence that functions in plant cellsto cause the termination of transcription and the addition ofpolyadenylated ribonucleotides to the 3′ end of the mRNA sequence.

The present invention also provides a transformed plant cell containinga nucleic acid molecule whose non-transcribed strand encodes a proteinor fragment thereof, wherein the transcribed strand of said nucleic acidis complementary to a nucleic acid molecule that encodes a protein orfragment thereof expressed in an effective amount to produce a desirableagronomic effect.

The present invention also provides bacterial, viral, microbial, andplant cells comprising the above mentioned elements (a), (b) and (c).

The present invention also provides a method of producing a plantcontaining one or more proteins encoded by sequences comprising SEQ IDNO:1 or complement thereof through SEQ ID NO:5521 or complement thereof,expressed in a sufficient amount and/or fashion to produce a desirableagronomic effect.

In accomplishing the foregoing, there is provided, in accordance withone aspect of the present invention, methods of producing geneticallytransformed plants, comprising the steps of:

-   -   (a) inserting into the genome of a plant cell a recombinant,        double-stranded DNA molecule comprising        -   (i) a promoter which functions in plant cells to cause the            production of an RNA sequence,        -   (ii) a structural DNA sequence that causes the production of            an RNA sequence which encodes a desired protein.        -   (iii) a 3′ non-translated DNA sequence which functions in            plant cells to cause the addition of polyadenylated            nucleotides to the 3′ end of RNA sequence; where the            promoter is homologous or heterologous with respect to the            coding sequence and adapted to cause sufficient expression            of a protein in desired plant tissues to enhance the            agronomic utility of a plant transformed with said gene.    -   (b) obtaining a transformed plant cell with said nucleic acid        molecule that encodes one or more proteins, wherein said nucleic        acid molecule is transcribed and results in expression of said        protein(s); and    -   (c) regenerating from the transformed plant cell a genetically        transformed plant

The present invention also encompasses differentiated plants, seeds, andprogeny comprising said transformed plant cells and which exhibit novelproperties of agronomic significance.

The present invention also provides a method of producing a plantcontaining reduced levels of a protein comprising: (A) transforming aplant cell with a nucleic acid molecule that encodes a protein, whereinsaid nucleic acid molecule is transcribed and results in co-suppressionof endogenous protein synthesis activity, and (B) regenerating plantsand producing subsequent progeny from the said transformed plant.

The present invention also provides a method of determining anassociation between a polymorphism and a plant trait comprising: (A)hybridizing a nucleic acid molecule specific for a polymorphism togenetic material of a plant, wherein said nucleic acid molecule isselected from the group consisting of SEQ ID NO: 1 through SEQ IDNO:5521; and (B) calculating the degree of association between thepolymorphism and the plant trait.

The present invention also provides a method of isolating a geneticregion, or nucleic acid that encodes a protein, peptide, or fragmentthereof comprising: (A) incubating under conditions permitting nucleicacid hybridization: a marker nucleic acid molecule, preferably an EST,with a complementary nucleic acid molecule obtained from a plant cell orplant tissue; (B) permitting hybridization between said marker nucleicacid molecule, preferably an EST, and said complementary nucleic acidmolecule obtained from said plant cell or plant tissue; and (C)isolating said complementary nucleic acid molecule.

The present invention also provides a method for determining a level orpattern in a plant cell of a protein in a plant comprising: (A)incubating, under conditions permitting nucleic acid hybridization, amarker nucleic acid molecule, the marker nucleic acid molecule selectedfrom the group of marker nucleic acid molecules which specificallyhybridize to a nucleic acid molecule having the nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1 through SEQ ID NO:5521 or complement thereof, with a complementary nucleic acid moleculeobtained from the plant cell or plant tissue, wherein nucleic acidhybridization between the marker nucleic acid molecule and thecomplementary nucleic acid molecule obtained from the plant cell orplant tissue permits the detection of an mRNA for the enzyme; (B)permitting hybridization between the marker nucleic acid molecule andthe complementary nucleic acid molecule obtained from the plant cell orplant tissue; and (C) detecting the level or pattern of thecomplementary nucleic acid, wherein the detection of the complementarynucleic acid is predictive of the level or pattern of the protein.

The present invention also provides a method for determining the levelor pattern of protein synthesis in a plant cell or plant tissuecomprising: (A) incubating under conditions permitting nucleic acidhybridization: a marker nucleic acid molecule, the marker nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of a nucleic acid molecule comprising SEQ ID NO: 1 orcomplement thereof through SEQ ID NO:5521 or complement thereof, with acomplementary nucleic acid molecule obtained from a plant cell or planttissue, wherein nucleic acid hybridization between the marker nucleicacid molecule, and the complementary nucleic acid molecule obtained fromthe plant cell or plant tissue permits the detection of a particularprotein synthesis; (B) permitting hybridization between the markernucleic acid molecule and the complementary nucleic acid moleculeobtained from the plant cell or plant tissue; and (C) detecting thelevel or pattern of the complementary nucleic acid, wherein thedetection of said complementary nucleic acid is predictive of the levelor pattern of the protein synthesis.

The present invention also provides a method for determining a level orpattern of protein synthesis in a plant cell or plant tissue underevaluation for suspected protein synthesis which comprises assaying theconcentration of a molecule, whose concentration is dependent upon theexpression of a gene, the gene having a nucleic acid sequence whichspecifically hybridizes to a protein synthesis marker nucleic acidmolecule, the molecule being present in a plant cell or plant tissue, incomparison to the concentration of that molecule present in a plant cellor plant tissue with a known level or pattern of protein synthesis,wherein an assayed concentration of the molecule is compared to theassayed concentration of the molecule in a plant cell or plant tissuewith a known level or pattern of protein synthesis.

The present invention also provides a method of determining a mutationin a plant whose presence is predictive of a mutation affecting a levelor pattern of a protein comprising the steps: (A) incubating, underconditions permitting nucleic acid hybridization, a marker nucleic acid,the marker nucleic acid selected from the group of marker nucleic acidmolecules which specifically hybridize to a nucleic acid moleculeconsisting of the nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1 through SEQ ID NO: 5521 or complement thereofand a complementary nucleic acid molecule obtained from the plant,wherein nucleic acid hybridization between the marker nucleic acidmolecule and the complementary nucleic acid molecule obtained from theplant permits the detection of a polymorphism whose presence ispredictive of a mutation affecting the level or pattern of the proteinin the plant; (B) permitting hybridization between the marker nucleicacid molecule and the complementary nucleic acid molecule obtained fromthe plant; and (C) detecting the presence of the polymorphism, whereinthe detection of the polymorphism is predictive of the mutation.

The present invention also provides a method for determining a mutationin a plant whose presence is predictive of a mutation affecting thelevel or pattern of protein synthesis comprising the steps: (A)incubating under conditions permitting nucleic acid hybridization: amarker nucleic acid molecule, the marker nucleic acid moleculecomprising a nucleic acid molecule that is linked to gene, the genehaving a nucleic acid sequence which specifically hybridizes to asequence selected from the group consisting of SEQ ID NO: 1 through SEQID NO:5521 and complements thereof and which encodes for a protein, anda complementary nucleic acid molecule obtained from a plant tissue orplant cell of the plant, wherein nucleic acid hybridization between themarker nucleic acid molecule and the complementary nucleic acid moleculeobtained from the plant permits the detection of a polymorphism whosepresence is predictive of a mutation affecting said level or pattern ofa protein synthesis in the plant; (B) permitting hybridization betweensaid marker nucleic acid molecule and said complementary nucleic acidmolecule obtained from said plant; and; (C) detecting the presence ofthe polymorphism, wherein the detection of the polymorphism ispredictive of the mutation.

The present invention also provides a method for reducing expression ofa protein in a plant cell, the method comprising: growing a transformedplant cell containing a nucleic acid molecule whose non-transcribedstrand encodes a protein or fragment thereof, wherein the transcribedstrand of said nucleic acid is complementary to a nucleic acid moleculethat encodes the protein in said plant cell, and whereby the strand thatis complementary to the nucleic acid molecule that encodes the proteinreduces or depresses expression of the protein.

Thus, the present invention provides soybean ESTs for use as moleculartags to isolate genetic regions (i.e. promoters and flanking sequences),isolate genes, map genes, and determine gene function. The presentinvention further provides soybean ESTs for use in determining if genesare members of a particular gene family.

The present invention also provides a method of obtaining full lengthgenes using soybean ESTs.

The present invention also provides a method of isolating promoters andflanking sequences using soybean ESTs.

The present invention also provides soybean ESTs for use inmarker-assisted breeding programs.

The present invention also provides a method of identifying tissuescomprising hybridizing nucleic acids from the tissue with soybean ESTs

The present invention also provides a method for production ofantibodies targeted against the proteins, peptides, or fragmentsproduced by the disclosed ESTs or variants or derivatives thereof.

The present invention also provides a method for the transformation andregeneration of plants comprising sequences hybridizable to thedisclosed ESTs.

The present invention also provides a method of modifying plant proteinexpression by inserting in a chimeric gene sense or antisense constructsof the soybean ESTs.

DETAILED DESCRIPTION OF THE INVENTION Agents

(a) Nucleic Acid Molecules

Agents of the present invention include nucleic acid molecules and morespecifically EST nucleic acid molecules or nucleic acid fragmentmolecules thereof. Fragment EST nucleic acid molecules may encodesignificant portion(s) of, or indeed most of, the EST nucleic acidmolecule. Alternatively, the fragments may comprise smalleroligonucleotides (having from about 15 to about 250 nucleotide residues,and more preferably, about 15 to about 30 nucleotide residues).

As used herein, an agent, be it a naturally occurring molecule orotherwise may be “substantially purified,” if desired, such that one ormore molecules that is or may be present in a naturally occurringpreparation containing that molecule will have been removed or will bepresent at a lower concentration than that at which it would normally befound.

The agents of the present invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic, and thus involve the capacity of the agentto mediate a chemical reaction or response.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g. DNA, peptide etc.),that is, or results, however indirect, from human manipulation of anucleic acid molecule.

It is understood that the agents of the present invention may be labeledwith reagents that facilitate detection of the agent (e.g. fluorescentlabels (Prober, et al., Science 238:336-340 (1987); Albarella et al., EP144914, chemical labels (Sheldon et al., U.S. Pat. No. 4,582,789;Albarella et al., U.S. Pat. No. 4,563,417, modified bases (Miyoshi etal., EP 119448, all of which are hereby incorporated by reference intheir entirety).

It is further understood, that the present invention provides bacterial,viral, microbial, and plant cells comprising the agents of the presentinvention.

EST nucleic acid molecules or fragment EST nucleic acid molecules arecapable of specifically hybridizing to other nucleic acid moleculesunder certain circumstances. As used herein, two nucleic acid moleculesare said to be capable of specifically hybridizing to one another if thetwo molecules are capable of forming an anti-parallel, double-strandednucleic acid structure. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule if they exhibit completecomplementarity. As used herein, molecules are said to exhibit “completecomplementarity” when every nucleotide of one of the molecules iscomplementary to a nucleotide of the other. Two molecules are said to be“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are said to be “complementary” if they can hybridize to oneanother with sufficient stability to permit them to remain annealed toone another under conventional “high-stringency” conditions.Conventional stringency conditions are described by Sambrook, et al.,In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1989), and by Haymes, et al. In:Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C. (1985), the entirety of which is herein incorporated by reference.Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. Thus, in order for an ESTnucleic acid molecule or fragment EST nucleic acid molecule to serve asa primer or probe it need only be sufficiently complementary in sequenceto be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

Appropriate stringency conditions which promote DNA hybridization are,for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or either the temperature or the salt concentration may be heldconstant while the other variable is changed.

In a preferred embodiment, a nucleic acid of the present invention willspecifically hybridize to one or more of the nucleic acid molecules setforth in SEQ ID NO: 1 through SEQ ID NO: 5521 or complements thereofunder moderately stringent conditions, for example at about 2.0×SSC andabout 65° C.

In a particularly preferred embodiment, a nucleic acid of the presentinvention will include those nucleic acid molecules that specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NO: 1 through SEQ ID NO: 5521 or complements thereof under highstringency conditions.

In one aspect of the present invention, the nucleic acid molecules ofthe present invention have one or more of the nucleic acid sequences setforth in SEQ ID NO: 1 through to SEQ ID NO:5521 or complements thereof.In another aspect of the present invention, one or more of the nucleicacid molecules of the present invention share between 100% and 90%sequence identity with one or more of the nucleic acid sequences setforth in SEQ ID NO: 1 through to SEQ ID NO:5521 or complements thereof.In a further aspect of the present invention, one or more of the nucleicacid molecules of the present invention share between 100% and 95%sequence identity with one or more of the nucleic acid sequences setforth in SEQ ID NO: 1 through to SEQ ID NO:5521 or complements thereof.In a more preferred aspect of the present invention, one or more of thenucleic acid molecules of the present invention share between 100% and98% sequence identity with one or more of the nucleic acid sequences setforth in SEQ ID NO: 1 through to SEQ ID NO:5521 or complements thereof.In an even more preferred aspect of the present invention, one or moreof the nucleic acid molecules of the present invention share between100% and 99% sequence identity with one or more of the sequences setforth in SEQ ID NO: 1 through to SEQ ID NO:5521 or complements thereof.In a further, even more preferred aspect of the present invention, oneor more of the nucleic acid molecules of the present invention exhibit100% sequence identity with one or more nucleic acid molecules presentwithin the cDNA library SOYMON001, herein designated SOYMON001 (MonsantoCompany, St. Louis, Mo., United States of America).

The degeneracy of the genetic code, which allows different nucleic acidsequences to code for the same protein or peptide, is known in theliterature. (U.S. Pat. No. 4,757,006, the entirety of which is hereinincorporated by reference). As used herein a nucleic acid molecule isdegenerate of another nucleic acid molecule when the nucleic acidmolecules encode for the same amino acid sequences but comprisedifferent nucleotide sequences. An aspect of the present invention isthat the nucleic acid molecules of the present invention include nucleicacid molecules that are degenerate of those set forth in SEQ ID NO: 1through to SEQ ID NO:5521 or complements thereof. As used herein,nucleic acid molecules are said to be capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure underconditions (e.g. salt and temperature) that permit hybridization ofsequences that exhibit 90% sequence identity or greater with each otherand exhibit this identity for at least a contiguous 50 base pairs of thenucleic acid molecules.

(b) Protein and Peptide Molecules

A class of agents comprises one or more of the protein or peptidemolecules encoded by SEQ ID NO: 1 through SEQ ID NO:5521 or complementsthereof or one or more of the protein or fragment thereof or peptidemolecules encoded by other nucleic acid agents of the present invention.As used herein, the term “protein molecule” or “peptide molecule”includes any molecule that comprises five or more amino acids. It iswell know in the art that proteins may undergo modification, includingpost-translational modifications, such as, but not limited to, disulfidebond formation, glycosylation, phosphorylation, or oligomerization.Thus, as used herein, the term “protein molecule” or “peptide molecule”includes any protein molecule that is modified by any biological ornon-biological process. The terms “amino acid” and “amino acids” referto all naturally occurring L-amino acids. This definition is meant toinclude norleucine, ornithine, homocysteine, and homoserine.

One or more of the protein or fragment of peptide molecules may beproduced via chemical synthesis, or more preferably, by expressing in asuitable bacterial or eukaryotic host. Suitable methods for expressionare described by Sambrook, et al., (In: Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989)), or similar texts.

A “protein fragment” is a peptide or polypeptide molecule whose aminoacid sequence comprises a subset of the amino acid sequence of thatprotein. A protein or fragment thereof that comprises one or moreadditional peptide regions not derived from that protein is a “fusion”protein. Such molecules may be derivatized to contain carbohydrate orother moieties (such as keyhole limpet hemocyanin, etc.). Fusion proteinor peptide molecule of the present invention are preferably produced viarecombinant means.

Another class of agents comprise protein or peptide molecules encoded bySEQ ID NO: 1 through SEQ ID NO:5521 or complements thereof or, fragmentsor fusions thereof in which non-essential, or not relevant, amino acidresidues have been added, replaced, or deleted. An example of such ahomologue is the homologue protein of all non-soybean plant species,including but not limited to alfalfa, Arabidopsis, barley, Brassica,broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape, onion,canola, flax, maize, an ornamental plant, pea, peanut, pepper, potato,rice, rye, sorghum, strawberry, sugarcane, sugarbeet, tomato, wheat,poplar, pine, fir, eukalyptus, apple, lettuce, peas, lentils, grape,banana, tea, turf grasses, etc. Particularly preferred non-soybeanplants to utilize for the isolation of homologues would include alfalfa,Arabidopsis, barley, cotton, corn, oat, oilseed rape, rice, corn,canola, ornamentals, sugarcane, sugarbeet, tomato, potato, wheat, andturf grasses. Such a homologue can be obtained by any of a variety ofmethods. Most preferably, as indicated above, one or more of thedisclosed sequences (SEQ ID NO: 1 through SEQ ID NO:5521 or complementsthereof) will be used to define a pair of primers that may be used toisolate the homologue-encoding nucleic acid molecules from any desiredspecies. Such molecules can be expressed to yield homologues byrecombinant means.

(c) Antibodies

One aspect of the present invention concerns antibodies, single-chainantigen binding molecules, or other proteins that specifically bind toone or more of the protein or peptide molecules of the present inventionand their homologues, fusions or fragments. Such antibodies may be usedto quantitatively or qualitatively detect the protein or peptidemolecules of the present invention. As used herein, an antibody orpeptide is said to “specifically bind” to a protein or peptide moleculeof the present invention if such binding is not competitively inhibitedby the presence of non-related molecules.

Nucleic acid molecules that encode all or part of the protein of thepresent invention can be expressed, via recombinant means, to yieldprotein or peptides that can in turn be used to elicit antibodies thatare capable of binding the expressed protein or peptide. Such antibodiesmay be used in immunoassays for that protein. Such protein-encodingmolecules, or their fragments may be a “fusion” molecule (i.e., a partof a larger nucleic acid molecule) such that, upon expression, a fusionprotein is produced. It is understood that any of the nucleic acidmolecules of the present invention may be expressed, via recombinantmeans, to yield proteins or peptides encoded by these nucleic acidmolecules.

The antibodies that specifically bind proteins and protein fragments ofthe present invention may be polyclonal or monoclonal, and may compriseintact immunoglobulins, or antigen binding portions of immunoglobulins(such as (F(ab′), F(ab′)₂) fragments, or single-chain immunoglobulinsproducible, for example, via recombinant means). It is understood thatpractitioners are familiar with the standard resource materials whichdescribe specific conditions and procedures for the construction,manipulation and isolation of antibodies (see, for example, Harlow andLane, In Antibodies: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1988), the entirety of which is herein incorporatedby reference).

Murine monoclonal antibodies are particularly preferred. BALB/c mice arepreferred for this purpose, however, equivalent strains may also beused. The animals are preferably immunized with approximately 25 μg ofpurified protein (or fragment thereof) that has been emulsified asuitable adjuvant (such as TiterMax adjuvant (Vaxcel, Norcross, Ga.)).Immunization is preferably conducted at two intramuscular sites, oneintraperitoneal site, and one subcutaneous site at the base of the tail.An additional i.v. injection of approximately 25 μg of antigen ispreferably given in normal saline three weeks later. After approximately11 days following the second injection, the mice may be bled and theblood screened for the presence of anti-protein or peptide antibodies.Preferably, a direct binding Enzyme-Linked Immunoassay (ELISA) isemployed for this purpose.

More preferably, the mouse having the highest antibody titer is given athird i.v. injection of approximately 25 μg of the same protein orfragment. The splenic leukocytes from this animal may be recovered 3days later, and are then permitted to fuse, most preferably, usingpolyethylene glycol, with cells of a suitable myeloma cell line (suchas, for example, the P3X63Ag8.653 myeloma cell line). Hybridoma cellsare selected by culturing the cells under “HAT”(hypoxanthine-aminopterin-thymine) selection for about one week. Theresulting clones may then be screened for their capacity to producemonoclonal antibodies (“mAbs), preferably by direct ELISA.

In one embodiment, anti-protein or peptide monoclonal antibodies areisolated using a fusion of a protein, protein fragment, or peptide ofthe present invention, or conjugate of a protein, protein fragment, orpeptide of the present invention, as immunogens. Thus, for example, agroup of mice can be immunized using a fusion protein emulsified inFreund's complete adjuvant (e.g. approximately 50 μg of antigen perimmunization). At three week intervals, an identical amount of antigenis emulsified in Freund's incomplete adjuvant and used to immunize theanimals. Ten days following the third immunization, serum samples aretaken and evaluated for the presence of antibody. If antibody titers aretoo low, a fourth booster can be employed. Polysera capable of bindingthe protein or peptide can also be obtained using this method.

In a preferred procedure for obtaining monoclonal antibodies, thespleens of the above-described immunized mice are removed, disrupted,and immune splenocytes are isolated over a ficoll gradient. The isolatedsplenocytes are fused, using polyethylene glycol with BALB/c-derivedHGPRT (hypoxanthine guanine phosphoribosyl transferase) deficientP3x63xAg8.653 plasmacytoma cells. The fused cells are plated into96-well microtiter plates and screened for hybridoma fusion cells bytheir capacity to grow in culture medium supplemented withhypothanthine, aminopterin and thymidine for approximately 2-3 weeks.

Hybridoma cells that arise from such incubation are preferably screenedfor their capacity to produce an immunoglobulin that binds to a proteinof interest. An indirect ELISA may be used for this purpose. In brief,the supernatants of hybridomas are incubated in microtiter wells thatcontain immobilized protein. After washing, the titer of boundimmunoglobulin can be determined using, for example, a goat anti-mouseantibody conjugated to horseradish peroxidase. After additional washing,the amount of immobilized enzyme is determined (for example through theuse of a chromogenic substrate). Such screening is performed as quicklyas possible after the identification of the hybridoma in order to ensurethat a desired clone is not overgrown by non-secreting neighbors.Desirably, the fusion plates are screened several times since the ratesof hybridoma growth vary. In a preferred sub-embodiment, a differentantigenic form of immunogen may be used to screen the hybridoma. Thus,for example, the splenocytes may be immunized with one immunogen, butthe resulting hybridomas can be screened using a different immunogen. Itis understood that any of the protein or peptide molecules of thepresent invention may be used to raise antibodies.

As discussed below, such antibody molecules or their fragments may beused for diagnostic purposes. Where the antibodies are intended fordiagnostic purposes, it may be desirable to derivatize them, for examplewith a ligand group (such as biotin) or a detectable marker group (suchas a fluorescent group, a radioisotope or an enzyme).

The ability to produce antibodies that bind the protein or peptidemolecules of the present invention permits the identification of mimeticcompounds of those molecules. A “mimetic compound” is a compound that isnot that compound, or a fragment of that compound, but which nonethelessexhibits an ability to specifically bind to antibodies directed againstthat compound.

It is understood that any of the agents of the present invention can besubstantially purified and/or be biologically active and/or recombinant.

Uses of the Agents of the Invention

The nucleic acid molecules and fragments thereof of the presentinvention were isolated from soybean leaf tissue. Leaves are thecarbohydrate factories of crop plants, therefore, the ESTs of thepresent invention will find great use in the isolation of a variety ofagronomically significant genes, including but not limited to genes thatare necessary for the interception and transformation of light energyvia photosynthesis linked with plant growth, quality, and yield. Genesisolated utilizing the disclosed ESTs would also be critical in pathwaysincluding but not limited to a pathway such as nitrogen metabolismlinked to fruiting and mobilization and distribution of nitrogen.

Nucleic acid molecules and fragments thereof of the present inventionmay be employed to obtain other nucleic acid molecules. Such moleculesinclude the nucleic acid molecule of other plants or other organisms(e.g., alfalfa, rice, potato, cotton, oat, rye, barley, maize, wheat,Arabidopsis, Brassica, etc.) including the nucleic acid molecules thatencode, in whole or in part, protein homologues of other plant speciesor other organisms, and sequences of genetic elements such as promotersand transcriptional regulatory elements. Such molecules can be readilyobtained by using the above-described nucleic acid molecules orfragments thereof to screen cDNA or genomic libraries obtained from suchplant species. Methods for forming such libraries are well known in theart. Such homologue molecules may differ in their nucleotide sequencesfrom those found in one or more of SEQ ID NO:1 through SEQ ID NO:5521 orcomplements thereof because complete complementarity is not needed forstable hybridization. The nucleic acid molecules of the presentinvention therefore also include molecules that, although capable ofspecifically hybridizing with the nucleic acid molecules may lack“complete complementarity.”

Any of a variety of methods may be used to obtain one or more of theabove-described nucleic acid molecules (Zamechik et al., Proc. Natl.Acad. Sci. (U.S.A.) 83:4143-4146 (1986), the entirety of which is hereinincorporated by reference; Goodchild et al., Proc. Natl. Acad. Sci.(U.S.A.) 85:5507-5511 (1988), the entirety of which is hereinincorporated by reference; Wickstrom et al., Proc. Natl. Acad. Sci.(U.S.A.) 85:1028-1032 (1988), the entirety of which is hereinincorporated by reference; Holt, et al., Molec. Cell. Biol. 8:963-973(1988), the entirety of which is herein incorporated by reference;Gerwirtz, et al., Science 242:1303-1306 (1988), the entirety of which isherein incorporated by reference); Anfossi, et al., Proc. Natl. Acad.Sci. (U.S.A.) 86:3379-3383 (1989), the entirety of which is hereinincorporated by reference; Becker, et al., EMBO J. 8:3685-3691 (1989);the entirety of which is herein incorporated by reference). Automatednucleic acid synthesizers may be employed for this purpose. In lieu ofsuch synthesis, the disclosed nucleic acid molecules may be used todefine a pair of primers that can be used with the polymerase chainreaction (Mullis, et al., Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); Erlich et al., EP 50,424; EP 84,796, EP 258,017, EP237,362; Mullis, EP 201,184; Mullis et al., U.S. Pat. No. 4,683,202;Erlich, U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No.4,683,194, all of which are hereby incorporated by reference in theirentirety) to amplify and obtain any desired nucleic acid molecule orfragment.

Promoter sequence(s) and other genetic elements including but notlimited to transcriptional regulatory elements associated with one ormore of the disclosed nucleic acid sequences can also be obtained usingthe disclosed nucleic acid sequences provided herein. In one embodiment,such sequences are obtained by incubating EST nucleic acid molecules orpreferably fragments thereof with members of genomic libraries (e.g. Zeamays and soybean) and recovering clones that hybridize to the ESTnucleic acid molecule or fragment thereof. In a second embodiment,methods of “chromosome walking,” or inverse PCR may be used to obtainsuch sequences (Frohman, et al., Proc. Natl. Acad. Sci. (U.S.A.)85:8998-9002 (1988); Ohara, et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:5673-5677 (1989); Pang et al., Biotechniques, 22(6); 1046-1048 (1977);Huang et al., Methods Mol. Biol. 69: 89-96 (1977); Hartl et al., MethodsMol. Biol. 58: 293-301 (1996), all of which are hereby incorporated byreference in their entirety). In one embodiment, the disclosed ESTs areused to identify cDNAs whose analogous genes contain promoters withdesirable expression patterns. The ESTs isolated from the library of thepresent invention are used to isolate promoters of tissue-enhanced,tissue-specific, developmentally- or environmentally-regulatedexpression profiles. Isolation and functional analysis of the 5′flanking promoter sequences of these genes from genomic libraries, forexample, using genomic screening methods and PCR techniques would resultin the isolation of useful promoters and transcriptional regulatoryelements. These methods are known to those of skill in the art and havebeen described (See for example Birren et al., Genome Analysis:Analyzing DNA, 1, (1997), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., the entirety of which is herein incorporated byreference). Promoters obtained utilizing the ESTs of the presentinvention could also be modified to affect their controlcharacteristics. Examples of such modifications would include but arenot limited to enhancer sequences as reported by Kay et al., Science236:1299 (1987), herein incorporated by reference in its entirety. Suchgenetic elements could be used to enhance gene expression of new andexisting traits for crop improvements.

The nucleic acid molecules of the present invention may be used toisolate promoters of tissue enhanced. tissue specific, cell-specific,cell-type, developmentally or environmentally regulated expressionprofiles. Isolation and functional analysis of the 5′ flanking promotersequences of these genes from genomic libraries, for example, usinggenomic screening methods and PCR techniques would result in theisolation of useful promoters and transcriptional regulatory elements.These methods are known to those of skill in the art and have beendescribed (See, for example, Birren et. al., Genome Analysis: AnalyzingDNA, 1, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1997), the entirety of which is herein incorporated by reference.).Promoters obtained utilizing the nucleic acid molecules of the presentinvention could also be modified to affect their controlcharacteristics. Examples of such modifications would include but arenot limited to enhancer sequences as reported by Kay, et al Science236:1299 (1987), herein incorporated reference in its entirety. Suchgenetic elements could be used to enhance gene expression of new andexisting traits for crop improvements.

In an aspect of the present invention, one or more of the nucleicmolecules of the present invention are used to determine whether a plant(preferably soybean) has a mutation affecting the level (i.e., theconcentration of mRNA in a sample, etc.) or pattern (i.e., the kineticsof expression, rate of decomposition, stability profile, etc.) of theexpression encoded in part or whole by one or more of the nucleic acidmolecule of the present invention (collectively, the “ExpressionResponse” of a cell or tissue). As used herein, the Expression Responsemanifested by a cell or tissue is said to be “altered” if it differsfrom the Expression Response of cells or tissues of plants notexhibiting the phenotype. To determine whether a Expression Response isaltered, the Expression Response manifested by the cell or tissue of theplant exhibiting the phenotype is compared with that of a similar cellor tissue sample of a plant not exhibiting the phenotype. As will beappreciated, it is not necessary to re-determine the Expression Responseof the cell or tissue sample of plants not exhibiting the phenotype eachtime such a comparison is made; rather, the Expression Response of aparticular plant may be compared with previously obtained values ofnormal plants. As used herein, the phenotype of the organism is any ofone or more characteristics of an organism (e.g. disease resistance,pest tolerance, environmental tolerance, male sterility, yield, qualityimprovements, etc.). A change in genotype or phenotype may be transientor permanent. Also as used herein, a tissue sample is any sample thatcomprises more than one cell. In a preferred aspect, a tissue samplecomprises cells that share a common characteristic (e.g. derived fromleaf, root, or pollen etc).

In one sub-aspect, such an analysis is conducted by determining thepresence and/or identity of polymorphism(s) by one or more of thenucleic acid molecules of the present invention and more specifically,one or more of the EST nucleic acid molecule or fragment thereof whichare associated with phenotype, or a predisposition to phenotype.

Any of a variety of molecules can be used to identify suchpolymorphism(s). In one embodiment, one or more of the EST nucleic acidmolecules (or a sub-fragment thereof) may be employed as a markernucleic acid molecule to identify such polymorphism(s). Alternatively,such polymorphisms can be detected through the use of a marker nucleicacid molecule or a marker protein that is genetically linked to (i.e., apolynucleotide that co-segregates with) such polymorphism(s).

In an alternative embodiment, such polymorphisms can be detected throughthe use of a marker nucleic acid molecule that is physically linked tosuch polymorphism(s). For this purpose, marker nucleic acid moleculescomprising a nucleotide sequence of a polynucleotide located within 1 mbof the polymorphism(s), and more preferably within 100 kb of thepolymorphism(s), and most preferably within 10 kb of the polymorphism(s)can be employed.

The genomes of animals and plants naturally undergo spontaneous mutationin the course of their continuing evolution (Gusella, Ann. Rev. Biochem.55:831-854 (1986)). A “polymorphism” is a variation or difference in thesequence of the gene or its flanking regions that arises in some of themembers of a species. The variant sequence and the “original” sequenceco-exist in the species' population. In some instances, suchco-existence is in stable or quasi-stable equilibrium.

A polymorphism is thus said to be “allelic,” in that, due to theexistence of the polymorphism, some members of a species may have theoriginal sequence (i.e., the original “allele”) whereas other membersmay have the variant sequence (i.e., the variant “allele”). In thesimplest case, only one variant sequence may exist, and the polymorphismis thus said to be di-allelic. In other cases, the species' populationmay contain multiple alleles, and the polymorphism is termedtri-allelic, etc. A single gene may have multiple different unrelatedpolymorphisms. For example, it may have a di-allelic polymorphism at onesite, and a multi-allelic polymorphism at another site.

The variation that defines the polymorphism may range from a singlenucleotide variation to the insertion or deletion of extended regionswithin a gene. In some cases, the DNA sequence variations are in regionsof the genome that are characterized by short tandem repeats (STRs) thatinclude tandem di- or tri-nucleotide repeated motifs of nucleotides.Polymorphisms characterized by such tandem repeats are referred to as“variable number tandem repeat” (“VNTR”) polymorphisms. VNTRs have beenused in identity analysis (Weber, U.S. Pat. No. 5,075,217; Armour, etal., FEBS Lett. 307:113-115 (1992); Jones, et al., Eur. J. Haematol.39:144-147 (1987); Horn, et al., PCT Application WO91/14003; Jeffreys,European Patent Application 370,719; Jeffreys, U.S. Pat. No. 5,699,082;Jeffreys. et al., Amer. J. Hum. Genet. 39:11-24 (1986); Jeffreys. etal., Nature 316:76-79 (1985); Gray, et al., Proc. R. Acad. Soc. Lond.243:241-253 (1991); Moore, et al., Genomics 10:654-660 (1991); Jeffreys,et al., Anim. Genet. 18:1-15 (1987); Hillel, et al., Anim. Genet.20:145-155 (1989); Hillel, et al., Genet. 124:783-789 (1990), all ofwhich are herein incorporated by reference in their entirety).

The detection of polymorphic sites in a sample of DNA may be facilitatedthrough the use of nucleic acid amplification methods. Such methodsspecifically increase the concentration of polynucleotides that span thepolymorphic site, or include that site and sequences located eitherdistal or proximal to it. Such amplified molecules can be readilydetected by gel electrophoresis or other means.

The most preferred method of achieving such amplification employs thepolymerase chain reaction (“PCR”) (Mullis, et al., Cold Spring HarborSymp. Quant. Biol. 51:263-273 (1986); Erlich, et al., European PatentAppln. 50,424; European Patent Appln. 84,796, European PatentApplication 258,017, European Patent Appln. 237,362; Mullis, EuropeanPatent Appln. 201,184; Mullis, et al., U.S. Pat. No. 4,683,202; Erlich.,U.S. Pat. No. 4,582,788; and Saiki, et al., U.S. Pat. No. 4,683,194, allof which are herein incorporated by reference), using primer pairs thatare capable of hybridizing to the proximal sequences that define apolymorphism in its double-stranded form.

In lieu of PCR, alternative methods, such as the “Ligase Chain Reaction”(“LCR”) may be used (Barany, Proc. Natl. Acad. Sci. (U.S.A.) 88:189-193(1991), the entirety of which is herein incorporated by reference. LCRuses two pairs of oligonucleotide probes to exponentially amplify aspecific target. The sequences of each pair of oligonucleotides isselected to permit the pair to hybridize to abutting sequences of thesame strand of the target. Such hybridization forms a substrate for atemplate-dependent ligase. As with PCR, the resulting products thusserve as a template in subsequent cycles and an exponentialamplification of the desired sequence is obtained.

LCR can be performed with oligonucleotides having the proximal anddistal sequences of the same strand of a polymorphic site. In oneembodiment, either oligonucleotide will be designed to include theactual polymorphic site of the polymorphism. In such an embodiment, thereaction conditions are selected such that the oligonucleotides can beligated together only if the target molecule either contains or lacksthe specific nucleotide that is complementary to the polymorphic sitepresent on the oligonucleotide. Alternatively, the oligonucleotides maybe selected such that they do not include the polymorphic site (see,Segev, PCT Application WO 90/01069, the entirety of which is hereinincorporated by reference).

The “Oligonucleotide Ligation Assay” (“OLA”) may alternatively beemployed (Landegren, et al., Science 241:1077-1080 (1988), the entiretyof which is herein incorporated by reference). The OLA protocol uses twooligonucleotides which are designed to be capable of hybridizing toabutting sequences of a single strand of a target. OLA, like LCR, isparticularly suited for the detection of point mutations. Unlike LCR,however, OLA results in “linear” rather than exponential amplificationof the target sequence.

Nickerson, et al. have described a nucleic acid detection assay thatcombines attributes of PCR and OLA (Nickerson, et al., Proc. Natl. Acad.Sci. (U.S.A.) 87:8923-8927 (1990), the entirety of which is hereinincorporated by reference). In this method, PCR is used to achieve theexponential amplification of target DNA, which is then detected usingOLA. In addition to requiring multiple, and separate, processing steps,one problem associated with such combinations is that they inherit allof the problems associated with PCR and OLA.

Schemes based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, arealso known (Wu, et al., Genomics 4:560 (1989), the entirety of which isherein incorporated by reference), and may be readily adapted to thepurposes of the present invention.

Other known nucleic acid amplification procedures, such asallele-specific oligomers, branched DNA technology, transcription-basedamplification systems, or isothermal amplification methods may also beused to amplify and analyze such polymorphisms (Malek, et al., U.S. Pat.No. 5,130,238; Davey, et al., European Patent Application 329,822;Schuster et al., U.S. Pat. No. 5,169,766; Miller, et al., PCTApplication WO 89/06700; Kwoh, et al., Proc. Natl. Acad. Sci. (U.S.A.)86:1173-1177 (1989); Gingeras, et al., PCT Application WO 88/10315;Walker, et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992), allof which are herein incorporated by reference in their entirety).

The identification of a polymorphism can be determined in a variety ofways. By correlating the presence or absence of it in an plant with thepresence or absence of a phenotype, it is possible to predict thephenotype of that plant. If a polymorphism creates or destroys arestriction endonuclease cleavage site, or if it results in the loss orinsertion of DNA (e.g., a VNTR polymorphism), it will alter the size orprofile of the DNA fragments that are generated by digestion with thatrestriction endonuclease. As such, individuals that possess a variantsequence can be distinguished from those having the original sequence byrestriction fragment analysis. Polymorphisms that can be identified inthis manner are termed “restriction fragment length polymorphisms”(“RFLPs”). RFLPs have been widely used in human and plant geneticanalyses (Glassberg, UK Patent Application 2135774; Skolnick, et al.,Cytogen. Cell Genet. 32:58-67 (1982); Botstein, et al., Ann. J. Hum.Genet. 32:314-331 (1980); Fischer, et al. (PCT Application WO90/13668);Uhlen, PCT Application WO90/11369).

Polymorphisms can also be identified by Single Strand ConformationPolymorphism (SSCP) analysis. The SSCP technique is a method capable ofidentifying most sequence variations in a single strand of DNA,typically between 150 and 250 nucleotides in length (Elles, Methods inMolecular Medicine: Molecular Diagnosis of Genetic Diseases, HumanaPress (1996), the entirety of which is herein incorporated byreference); Orita et al., Genomics 5: 874-879 (1989), the entirety ofwhich is herein incorporated by reference). Under denaturing conditionsa single strand of DNA will adopt a conformation that is uniquelydependent on its sequence conformation. This conformation usually willbe different, even if only a single base is changed. Most conformationshave been reported to alter the physical configuration or sizesufficiently to be detectable by electrophoresis. A number of protocolshave been described for SSCP including, but not limited to Lee et al.,Anal. Biochem. 205: 289-293 (1992), the entirety of which is hereinincorporated by reference; Suzuki et al., Anal. Biochem. 192: 82-84(1991), the entirety of which is herein incorporated by reference; Lo etal., Nucleic Acids Research 20: 1005-1009 (1992), the entirety of whichis herein incorporated by reference; Sarkar et al., Genomics 13: 441-443(1992), the entirety of which is herein incorporated by reference). Itis understood that one or more of the nucleic acids of the presentinvention, may be utilized as markers or probes to detect polymorphismsby SSCP analysis.

Polymorphisms may also be found using a DNA fingerprinting techniquecalled amplified fragment length polymorphism (AFLP), which is based onthe selective PCR amplification of restriction fragments from a totaldigest of genomic DNA to profile that DNA. Vos, et al., Nucleic AcidsRes. 23:4407-4414 (1995), the entirety of which is herein incorporatedby reference. This method allows for the specific co-amplification ofhigh numbers of restriction fragments, which can be visualized by PCRwithout knowledge of the nucleic acid sequence.

AFLP employs basically three steps. Initially, a sample of genomic DNAis cut with restriction enzymes and oligonucleotide adapters are ligatedto the restriction fragments of the DNA. The restriction fragments arethen amplified using PCR by using the adapter and restriction sequenceas target sites for primer annealing. The selective amplification isachieved by the use of primers that extend into the restrictionfragments, amplifying only those fragments in which the primerextensions match the nucleotide flanking the restriction sites. Theseamplified fragments are then visualized on a denaturing polyacrylamidegel.

AFLP analysis has been performed on Salix (Beismann, et al., Mol. Ecol.6:989-993 (1997), the entirety of which is herein incorporated byreference); Acinetobacter (Janssen, et al., Int. J. Syst. Bacteriol47:1179-1187 (1997), the entirety of which is herein incorporated byreference), Aeromonas popoffi (Huys, et al., Int. J. Syst. Bacteriol.47:1165-1171 (1997), the entirety of which is herein incorporated byreference), rice (McCouch, et al., Plant Mol. Biol. 35:89-99 (1997), theentirety of which is herein incorporated by reference); Nandi, et al.,Mol. Gen. Genet. 255:1-8 (1997); Cho, et al., Genome 39:373-378 (1996),herein incorporated by reference), barley (Hordeum vulgare)(Simons, etal., Genomics 44:61-70 (1997), the entirety of which is hereinincorporated by reference; Waugh, et al., Mol. Gen. Genet. 255:311-321(1997), the entirety of which is herein incorporated by reference; Qi,et al., Mol. Gen. Genet. 254:330-336 (1997), the entirety of which isherein incorporated by reference; Becker, et al., Mol. Gen. Genet.249:65-73 (1995), the entirety of which is herein incorporated byreference), potato (Van der Voort, et al., Mol. Gen. Genet. 255:438-447(1997), the entirety of which is herein incorporated by reference;Meksem, et al., Mol. Gen. Genet. 249:74-81 (1995), the entirety of whichis herein incorporated by reference), Phytophthora infestans (Van derLee, et al., Fungal Genet. Biol. 21:278-291 (1997), the entirety ofwhich is herein incorporated by reference), Bacillus anthracis (Keim, etal., J. Bacteriol. 179:818-824 (1997)), Astragalus cremnophylax (Travis,et al., Mol. Ecol. 5:735-745 (1996), the entirety of which is hereinincorporated by reference), Arabidopsis (Cnops, et al., Mol. Gen. Genet.253:3241 (1996), the entirety of which is herein incorporated byreference), Escherichia coli (Lin, et al., Nucleic Acids Res.24:3649-3650 (1996), the entirety of which is herein incorporated byreference), Aeromonas (Huys, et al., Int. J. Syst. Bacteriol. 46:572-580(1996), the entirety of which is herein incorporated by reference),nematode (Folkertsma, et al., Mol. Plant. Microbe Interact. 9:47-54(1996), the entirety of which is herein incorporated by reference),tomato (Thomas, et al., Plant J. 8:785-794 (1995), the entirety of whichis herein incorporated by reference), and human (Latorra, et al., PCRMethods Appl. 3:351-358 (1994)). AFLP analysis has also been used forfingerprinting mRNA (Money, et al., Nucleic Acids Res. 24:2616-2617(1996), the entirety of which is herein incorporated by reference;Bachem, et al., Plant J. 9:745-753 (1996), the entirety of which isherein incorporated by reference). It is understood that one or more ofthe nucleic acids of the present invention, may be utilized as markersor probes to detect polymorphisms by AFLP analysis for fingerprintingmRNA.

Polymorphisms may also be found using random amplified polymorphic DNA(RAPD) (Williams et al., Nucl. Acids Res. 18: 6531-6535 (1990), theentirety of which is herein incorporated by reference) and cleaveableamplified polymorphic sequences (CAPS) (Lyamichev et al., Science 260:778-783 (1993), the entirety of which is herein incorporated byreference). It is understood that one or more of the nucleic acids ofthe present invention, may be utilized as markers or probes to detectpolymorphisms by RAPD or CAPS analysis.

Polymorphisms are useful, through linkage analysis, to define thegenetic distances or physical distances between polymorphic traits. Aphysical map or ordered array of genomic DNA fragments in the desiredregion containing the gene may be used to characterize and isolate genescorresponding to desirable traits. For this purpose, yeast artificialchromosomes (YACs), bacterial artificial chromosomes (BACs), and cosmidsare appropriate vectors for cloning large segments of DNA molecules.Although fewer clones are needed to make a contig for a specific genomicregion by using YACs (Agyare et al., Genome Res. 7: 1-9 (1997), theentirety of which is herein incorporated by reference; James et al.,Genomics 32: 425-430 (1996), the entirety of which is hereinincorporated by reference), chimerism in the inserted DNA fragment canarise. Cosmids are convenient for handling smaller-size DNA moleculesand may be used for transformation in developing transgenic plants. BACsalso carry DNA fragments and are less prone to chimerism.

Through genetic mapping, a fine scale linkage map can be developed usingDNA markers, and, then, a genomic DNA library of large-sized fragmentscan be screened with molecular markers linked to the desired trait.Molecular markers are advantageous for agronomic traits that areotherwise difficult to tag, such as resistance to pathogens, insects andnematodes, tolerance to abiotic stresses, quality parameters andquantitative traits. The essential requirements for marker-assistedselection in a plant breeding program are: (1) the marker(s) shouldco-segregate or be closely linked with the desired trait; (2) anefficient means of screening large populations for the molecularmarker(s) should be available; and (3) the screening technique shouldhave high reproducibility across laboratories, be economical to use andbe user-friendly. Molecular marker studies using near-isogenic lines(NILs) (Martin et al., Proc. Natl. Acad. Sci. (U.S.A). 88: 2336-2340(1991), herein incorporate by reference; Young et al., Genetics 120:579-585. (1988), the entirety of which is herein incorporated byreference, bulked segment analysis (Michelmore et al., Proc. Natl. Acad.Sci. (U.S.A) 88: 9828-9832 (1991), the entirety of which is hereinincorporated by reference) or recombinant inbred lines (Mohan et al.,Theor. Appl. Genet. 87: 782-788 (1994), the entirety of which is hereinincorporated by reference) have been used to map genes indifferent plantspecies (Coe and Neuffer, In: Genetic maps: locus maps of complexgenomes, ed. S. J. O'Brien, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 157-189 (1993), the entirety of which is hereinincorporated by reference). It is understood that one or more of thenucleic acid molecules of the present invention may be used as molecularmarkers.

In accordance with this aspect of the present invention, a samplenucleic acid is obtained from plants cells or tissues. Any source ofnucleic acid may be used. Preferably, the nucleic acid is genomic DNA.The nucleic acid is subjected to restriction endonuclease digestion. Forexample, one or more EST nucleic acid molecule or fragment thereof canbe used as a probe in accordance with the above-described polymorphicmethods. The polymorphism obtained in this approach can then be clonedto identify the mutation at the coding region which alters the protein'sstructure or regulatory region of the gene which affects its expressionlevel.

In one aspect of the present invention, an evaluation can be conductedto determine whether a particular mRNA molecule is present. One or moreof the nucleic acid molecules of the present invention, preferably oneor more of the EST nucleic acid molecules of the present invention areutilized to detect the presence or quantity of the mRNA species. Suchmolecules are then incubated with cell or tissue extracts of a plantunder conditions sufficient to permit nucleic acid hybridization. Thedetection of double-stranded probe-mRNA hybrid molecules is indicativeof the presence of the mRNA; the amount of such hybrid formed isproportional to the amount of mRNA. Thus, such probes may be used toascertain the level and extent of the mRNA production in a plant's cellsor tissues. Such nucleic acid hybridization may be conducted underquantitative conditions (thereby providing a numerical value of theamount of the mRNA present). Alternatively, the assay may be conductedas a qualitative assay that indicates either that the mRNA is present,or that its level exceeds a user set, predefined value.

A principle of in situ hybridization is that a labeled, single-strandednucleic acid probe will hybridize to a complementary strand of cellularDNA or RNA and, under the appropriate conditions, these molecules willform a stable hybrid. When nucleic acid hybridization is combined withhistological techniques, specific DNA or RNA sequences can be identifiedwithin a single cell. An advantage of in situ hybridization over moreconventional techniques for the detection of nucleic acids is that itallows and investigator to determine the precise spatial population(Angerer et al., Dev. Biol. 101: 477-484 (1984), the entirety of whichis herein incorporated by reference; Angerer et al., Dev. Biol. 112:157-166 (1985), the entirety of which is herein incorporated byreference; Dixon et al., EMBO J. 10: 1317-1324 (1991), the entirety ofwhich is herein incorporated by reference). In situ hybridization may beused to measure the steady-state level of RNA accumulation. It is asensitive technique and RNA sequences present in as few as 5-10 copiesper cell can be detected (Hardin et al., J. Mol. Biol. 202: 417-431.(1989), the entirety of which is herein incorporated by reference). Anumber of protocols have been devised for in situ hybridization, eachwith tissue preparation, hybridization, and washing conditions(Meyerowitz, Plant Mol. Biol. Rep. 5: 242-250 (1987), the entirety ofwhich is herein incorporated by reference; Cox and Goldberg, In: PlantMolecular Biology: A Practical Approach (ed. C. H. Shaw), pp. 1-35. IRLPress, Oxford (1988), the entirety of which is herein incorporated byreference; Raikhel et al., In situ RNA hybridization in plant tissues.In Plant Molecular Biology Manual, vol. B9: 1-32. Kluwer AcademicPublisher, Dordrecht, Belgium (1989), the entirety of which is hereinincorporated by reference).

In situ hybridization also allows for the localization of proteinswithin a tissue or cell (Wilkinson, In Situ Hybridization, OxfordUniversity Press, Oxford (1992), the entirety of which is hereinincorporated by reference; Langdale, In Situ Hybridization 165-179 In:The Maize Handbook, eds. Freeling and Walbot, Springer-Verlag, New York(1994), the entirety of which is herein incorporated by reference). Itis understood that one or more of the molecules of the presentinvention, preferably one or more of the EST nucleic acid molecules ofthe present invention or one or more of the antibodies of the presentinvention may be utilized to detect the level or pattern of a cytokininpathway enzyme or fragment thereof by in situ hybridization.

Fluorescent in situ hybridization also enables the localization of aparticular DNA sequence along a chromosome which is useful, among otheruses, for gene mapping, following chromosomes in hybrid lines ordetecting chromosomes with translocations, transversions or deletions.In situ hybridization has been used to identify chromosomes in severalplant species (Griffor et al., Plant Mol. Biol. 17: 101-109 (1991), theentirety of which is herein incorporated by reference; Gustafson et al.,Proc. Nat'l Acad. Sci. (U.S.A.). 87: 1899-1902 (1990), hereinincorporated by reference; Mukai and Gill, Genome 34: 448-452. (1991);Schwarzacher and Heslop-Harrison, Genome 34: 317-323 (1991); Wang etal., Jpn. J. Genet. 66: 313-316 (1991), the entirety of which is hereinincorporated by reference; Parra and Windle, Nature Genetics, 5: 17-21(1993), the entirety of which is herein incorporated by reference). Itis understood that the nucleic acid molecules of the present inventionmay be used as probes or markers to localize sequences along achromosome

It is also understood that one or more of the molecules of the presentinvention, preferably one or more of the EST nucleic acid molecules ofthe present invention or one or more of the antibodies of the presentinvention may be utilized to detect the expression level or pattern of aprotein or mRNA thereof by in situ hybridization.

Another method to localize the expression of a molecule is tissueprinting. Tissue printing provides a way to screen, at the same time onthe same membrane many tissue sections from different plants ordifferent developmental stages. Tissue-printing procedures utilize filmsdesigned to immobilize proteins and nucleic acids. In essence, a freshlycut section of an organ is pressed gently onto nitrocellulose paper,nylon membrane or polyvinylidene difluoride membrane. Such membranes arecommercially available (e.g. Millipore, Bedford, Mass.). The contents ofthe cut cell transfer onto the membrane, and the molecules areimmobilized to the membrane. The immobilized molecules form a latentprint that can be visualized with appropriate probes. When a planttissue print is made on nitrocellulose paper, the cell walls leave aphysical print that makes the anatomy visible without further treatment(Vamer and Taylor, Plant Physiol. 91: 31-33 (1989), the entirety ofwhich is herein incorporated by reference).

Tissue printing on substrate films is described by Daoust, Exp. CellRes. 12: 203-211 (1957), the entirety of which is herein incorporated byreference, who detected amylase, protease, ribonuclease, anddeoxyribonuclease in animal tissues using starch, gelatin, and agarfilms. These techniques can be applied to plant tissues (Yomo andTaylor, Planta 112:35-43 (1973); Harris and Chrispeels, Plant Physiol.56: 292-299 (1975). Advances in membrane technology have increased therange of applications of Daoust's tissue-printing techniques allowing(Cassab and Varner, J. Cell. Biol. 105: 2581-2588 (1987), the entiretyof which is herein incorporated by reference; the histochemicallocalization of various plant enzymes and deoxyribonuclease onnitrocellulose paper and nylon (Spruce et al., Phytochemistry, 26:2901-2903 (1987), the entirety of which is herein incorporated byreference; Barres et al. Neuron 5: 527-544 (1990), the entirety of whichis herein incorporated by reference; the entirety of which is hereinincorporated by reference; Reid and Pont-Lezica, Tissue Printing: Toolsfor the Study of Anatomy, Histochemistry, and Gene Expression, AcademicPress, New York, N.Y. (1992), the entirety of which is hereinincorporated by reference; Reid et al. Plant Physiol. 93: 160-165(1990), herein incorporate by reference; Ye et al. Plant J. 1: 175-183(1991), the entirety of which is herein incorporated by reference).

It is understood that one or more of the molecules of the presentinvention, preferably one or more of the EST nucleic acid molecules ofthe present invention or one or more of the antibodies of the presentinvention may be utilized to detect the presence or quantity of aprotein by tissue printing.

Further, it is also understood that any of the nucleic acid molecules ofthe present invention may be used as marker nucleic acids and or probesin connection with methods that require probes or marker nucleic acids.As used herein, a probe is an agent that is utilized to determine anattribute or feature (e.g. presence or absence, location, correlation,etc.) or a molecule, cell, tissue or plant. As used herein, a markernucleic acid is a nucleic acid molecule that is utilized to determine anattribute or feature (e.g., presence or absence, location, correlation,etc.) or a molecule, cell, tissue or plant.

A microarray-based method for high-throughput monitoring of plant geneexpression may be utilized to measure gene-specific hybridizationtargets. This ‘chip’-based approach involves using microarrays ofnucleic acid molecules as gene-specific hybridization targets toquantitatively measure expression of the corresponding plant genes(Schena et al., Science 270: 467-470 (1995), the entirety of which isherein incorporated by reference; Shalon, Ph.D. Thesis. StanfordUniversity (1996), the entirety of which is herein incorporated byreference). Every nucleotide in a large sequence can be queried at thesame time. Hybridization can be used to efficiently analyze largeamounts of nucleotide sequence.

Several microarray methods have been described. One method compares thesequences to be analyzed by hybridization to a set of oligonucleotidesrepresenting all possible subsequences (Bains and Smith, J. Theor. Biol.135: 303 (1989), the entirety of which is herein incorporated byreference). A second method hybridizes the sample to an array ofoligonucleotide probes. An array consisting of oligonucleotidescomplementary to subsequences of a target sequence can be used todetermine the identity of a target sequence, measure its amount, anddetect differences between the target and a reference sequence. Nucleicacid molecules microarrays may also be screened with protein moleculesor fragments thereof to determine nucleic acid molecules thatspecifically bind protein molecules or fragments thereof.

The microarray approach may be used with polypeptide targets (U.S. Pat.No. 5,445,934; U.S. Pat. No. 5,143,854; U.S. Pat. No. 5,079,600; U.S.Pat. No. 4,923,901, all of which are herein incorporated by reference intheir entirety). Essentially, polypeptides are synthesized on asubstrate (microarray) and these polypeptides can be screened witheither protein molecules or fragments thereof or nucleic acid moleculesin order to screen for either protein molecules or fragments thereof ornucleic acid molecules that specifically bind the target polypeptides.Implementation of these techniques rely on recently developedcombinatorial technologies to generate any ordered array of a largenumber of oligonucleotide probes (Fodor et al., Science 251:767-773(1991), the entirety of which is herein incorporated by reference).

It is understood that one or more of the molecules of the presentinvention, preferably one or more of the nucleic acid molecules orprotein molecules or fragments thereof of the present invention may beutilized in a microarray based method.

Site-directed mutagenesis may be utilized to modify nucleic acidsequences, particularly as it is a technique that allows one or more ofthe amino acids encoded by a nucleic acid molecule to be altered (e.g. athreonine to be replaced by a methionine). Three basic methods forsite-directed mutagenesis are often employed. These are cassettemutagenesis (Wells et al., Gene 34:315-23 (1985), the entirety of whichis herein incorporated by reference), primer extension (Gilliam et al.,Gene 12:129-137 (1980), the entirety of which is herein incorporated byreference); Zoller and Smith, Methods Enzymol. 100:468-500 (1983), theentirety of which is herein incorporated by reference; andDalbadie-McFarland et al., Proc. Natl. Acad. Sci. (U.S.A.) 79:6409-6413(1982), the entirety of which is herein incorporated by reference) andmethods based upon PCR (Scharf et al., Science 233:1076-1078 (1986), theentirety of which is herein incorporated by reference; Higuchi et al.,Nucleic Acids Res. 16:7351-7367 (1988), the entirety of which is hereinincorporated by reference). Site-directed mutagenesis approaches arealso described in European Patent 0 385 962, the entirety of which isherein incorporated by reference, European Patent 0 359 472, theentirety of which is herein incorporated by reference, and PCT PatentApplication WO 93/07278, the entirety of which is herein incorporated byreference.

Site-directed mutagenesis strategies have been applied to plants forboth in vitro as well as in vivo site-directed mutagenesis (Lanz et al.,J. Biol. Chem. 266:9971-6 (1991), the entirety of which is hereinincorporated by reference; Kovgan and Zhdanov, Biotekhnologiya5:148-154, No. 207160n, Chemical Abstracts 110:225 (1989), the entiretyof which is herein incorporated by reference; Ge et al., Proc. Natl.Acad. Sci. (U.S.A.) 86:4037-4041 (1989), the entirety of which is hereinincorporated by reference, Zhu et al., J. Biol. Chem. 271:18494-18498(1996), Chu et al., Biochemistry 33:6150-6157 (1994), the entirety ofwhich is herein incorporated by reference, Small et al., EMBO J.11:1291-1296 (1992), the entirety of which is herein incorporated byreference, Cho et al., Mol. Biotechnol. 8:13-16 (1997), Kita et al., J.Biol. Chem. 271:26529-26535 (1996), the entirety of which is hereinincorporated by reference, Jin et al., Mol. Microbiol. 7:555-562 (1993),the entirety of which is herein incorporated by reference, Hatfield andVierstra, J. Biol. Chem. 267:14799-14803 (1992), the entirety of whichis herein incorporated by reference, Zhao et al., Biochemistry31:5093-5099 (1992), the entirety of which is herein incorporated byreference).

Any of the nucleic acid molecules of the present invention may either bemodified by site-directed mutagenesis or used as, for example, nucleicacid molecules that are used to target other nucleic acid molecules formodification. It is understood that mutants with more than one alterednucleotide can be constructed using techniques that practitionersskilled in the art are familiar with such as isolating restrictionfragments and ligating such fragments into an expression vector (see,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (1989)).

Sequence-specific DNA-binding proteins play a role in the regulation oftranscription. The isolation of recombinant cDNAs encoding theseproteins facilitates the biochemical analysis of their structural andfunctional properties. Genes encoding such DNA-binding proteins havebeen isolated using classical genetics (Vollbrecht et al., Nature 350:241-243 (1991), the entirety of which is herein incorporated byreference) and molecular biochemical approaches, including the screeningof recombinant cDNA libraries with antibodies (Landschulz et al., GenesDev. 2: 786-800 (1988), the entirety of which is herein incorporated byreference) or DNA probes (Bodner et al., Cell 55: 505-518 (1988), theentirety of which is herein incorporated by reference). In addition, anin situ screening procedure has been used and has facilitated theisolation of sequence-specific DNA-binding proteins from various plantspecies (Gilmartin et al., Plant Cell 4: 839-849 (1992), the entirety ofwhich is herein incorporated by reference; Schindler et al., EMBO J. 11:1261-1273 (1992) the entirety of which is herein incorporated byreference). An in situ screening protocol does not require thepurification of the protein of interest (Vinson et al., Genes Dev. 2:801-806 (1988), the entirety of which is herein incorporated byreference; Singh et al., Cell 52: 415-423 (1988), the entirety of whichis herein incorporated by reference).

Steps may be employed to characterize DNA-protein interactions. Thefirst is to identify promoter fragments that interact with DNA-bindingproteins, to titrate binding activity, to determine the specificity ofbinding, and to determine whether a given DNA-binding activity caninteract with related DNA sequences (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2^(nd) edition. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). Electrophoretic mobility-shiftassay is a widely used assay. The assay provides a simple, rapid, andsensitive method for detecting DNA-binding proteins based on theobservation that the mobility of a DNA fragment through a nondenaturing,low-ionic strength polyacrylamide gel is retarded upon association witha DNA-binding protein (Fried and Crother, Nucleic Acids Res. 9:6505-6525 (1981), the entirety of which is herein incorporated byreference). When one or more specific binding activities have beenidentified, the exact sequence of the DNA bound by the protein may bedetermined. Several procedures for characterizing protein/DNA-bindingsites are used, including methylation and ethylation interference assays(Maxam and Gilbert, Methods Enzymol. 65: 499-560 (1980), the entirety ofwhich is herein incorporated by reference; Wissman and hillen, MethodsEnzymol. 208: 365-379 (1991), the entirety of which is hereinincorporated by reference) and footprinting techniques employing DNase I(Galas and Schmitz, Nucleic Acids Res. 5: 3157-3170 (1978), the entiretyof which is herein incorporated by reference),1,10-phenanthroline-copper ion methods (Sigman et al., Methods Enzymol.208: 365-379 (1991), the entirety of which is herein incorporated byreference) or hydroxyl radical methods (Dixon et al., Methods Enzymol.208: 380-413 (1991), the entirety of which is herein incorporated byreference). It is understood that one or more of the nucleic acidmolecules of the present invention, preferably one or more of the ESTnucleic acid molecules of the present invention may be utilized toidentify a protein or fragment thereof that specifically binds to anucleic acid molecule of the present invention. It is also understoodthat one or more of the protein molecules or fragments thereof of thepresent invention may be utilized tp identify a nucleic acid moleculethat specifically binds to it.

The two-hybrid system is based on the fact that many cellular functionsare carried out by proteins that interact (physically) with one another.Two-hybrid systems have been used to probe the function of new proteins(Chien et al., Proc. Natl. Acad. Sci. (U.S.A.) 88: 9578-9582 (1991) theentirety of which is herein incorporated by reference; Durfee et al.,Genes Dev. 7: 555-569 (1993) the entirety of which is hereinincorporated by reference; Choi et al., Cell 78: 499-512 (1994), theentirety of which is herein incorporated by reference; Kranz et al.,Genes Dev. 8: 313-327 (1994), the entirety of which is hereinincorporated by reference).

Interaction mating techniques have facilitated a number of two-hybridstudies of protein-protein interaction. Interaction mating has been usedto examine interactions between small sets of tens of proteins (Finleyand Brent, Proc. Natl. Acad. Sci. (U.S.A.) 91: 12098-12984 (1994), theentirety of which is herein incorporated by reference), larger sets ofhundreds of proteins, (Bendixen et al., Nucl. Acids Res. 22: 1778-1779(1994), the entirety of which is herein incorporated by reference) andto comprehensively map proteins encoded by a small genome (Bartel etal., Nature Genetics 12: 72-77 (1996), the entirety of which is hereinincorporated by reference). This technique utilizes proteins fused tothe DNA-binding domain and proteins fused to the activation domain. Theyare expressed in two different haploid yeast strains of opposite matingtype, and the strains are mated to determine if the two proteinsinteract. Mating occurs when haploid yeast strains come into contact andresult in the fusion of the two haploids into a diploid yeast strain. Aninteraction can be determined by the activation of a two-hybrid reportergene in the diploid strain. The primary advantage of this technique isthat it reduces the number of yeast transformations needed to testindividual interactions. It is understood that the protein-proteininteractions of protein or fragments thereof of the present inventionmay be investigated using the two-hybrid system and that any of thenucleic acid molecules of the present invention that encode suchproteins or fragments thereof may be used to transform yeast in thetwo-hybrid system.

Synechocystis 6803 is a photosynthetic Cyanobacterium capable ofoxygenic photosynthesis as well as heterotrophic growth in the absenceof light. The entire genome has been sequenced, and it is reported tohave a circular genome size of 3.57 Mbp containing 3168 potential openreading frames. Open reading frames (ORFs) were identified based upontheir homology to other reported ORFs and by using ORF identificationcomputer programs. Sixteen hundred potential ORFs were assigned based ontheir homology to previously identified ORFs. Of these 1600 ORFs, 145were identical to reported ORFs (Kaneko et al., DNA Research 3:109-36(1996), herein incorporated by reference in its entirety).

Several prokaryote promoters have been used in Synechocystis to expressheterologous genes including the tac, lac, and lambda phage promoters(Bryant (ed.), The Molecular Biology of Cyanobacteria, Kluwer AcademicPublishers, (1994); Ferino and Chauvat, Gene 84:257-266 (1989), both ofwhich are herein incorporated by reference in their entirety). Severalbacterial origins of replication such as RSF1010 and ACYC are reportedto replicate in Synechocystis (Mermet-Bouvier and Chauvat, CurrentMicrobiology 28:145-148 (1994); Kuhlemeier et al., Mol. Gen. Genet.184:249-254 (1981), both of which are herein incorporated by referencein their entirety).

Synechocystis has been used to study gene regulation by gene replacementthrough homologous recombination or by gene disruption using antibioticresistance markers (Pakrasi et al., EMBO 7:325-332 (1988), hereinincorporated by reference in its entirety). In such gene regulationstudies, double reciprocal homologous regions of the host genomeflanking the gene of interest recombine to stably integrate the gene ofinterest into the genome. The gene of interest can be expressed oncethat gene has been stably integrated into the genome. Biochemicalanalysis can be performed to study the effect of the replaced or deletedgene.

It is understood that the agents of the present invention may beemployed in a Synechocystis system.

Exogenous genetic material may be transferred into a plant cell and theplant cell regenerated into a whole, fertile or sterile plant. Exogenousgenetic material is any genetic material, whether naturally occurring orotherwise, from any source that is capable of being inserted into anyorganism. Such genetic material may be transferred into eithermonocotyledons and dicotyledons including but not limited to the crops,Zea mays and soybean (See specifically, Chistou, Particle Bombardmentfor Genetic Engineering of Plants, pp 63-69 (zea mays), pp 50-60(soybean), Biotechnology Intelligence Unit. Academic Press, San Diego,Calif. (1996), the entirety of which is herein incorporated by referenceand generally Chistou, Particle Bombardment for Genetic Engineering ofPlants, Biotechnology Intelligence Unit. Academic Press, San Diego,Calif. (1996), the entirety of which is herein incorporated byreference).

Transfer of a nucleic acid that encodes for a protein can result inoverexpression of that protein in a transformed cell or transgenicplant. One or more of the proteins or fragments thereof encoded bynucleic acid molecules of the present invention may be overexpressed ina transformed cell or transformed plant. Such overexpression may be theresult of transient or stable transfer of the exogenous material.

Exogenous genetic material may be transferred into a plant cell by theuse of a DNA vector or construct designed for such a purpose. Design ofsuch a vector is generally within the skill of the art (See, PlantMolecular Biology: A Laboratory Manual eds. Clark, Springer, New York(1997), the entirety of which is herein incorporated by reference).

A construct or vector may include a plant promoter to express theprotein or protein fragment of choice. A number of promoters which areactive in plant cells have been described in the literature. Theseinclude the nopaline synthase (NOS) promoter (Ebert et al., Proc. Natl.Acad. Sci. (U.S.A.) 84:5745-5749 (1987), the entirety of which is hereinincorporated by reference), the octopine synthase (OCS) promoter (whichare carried on tumor-inducing plasmids of Agrobacterium tumefaciens),the caulimovirus promoters such as the cauliflower mosaic virus (CaMV)19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987), theentirety of which is herein incorporated by reference) and the CAMV 35Spromoter (Odell et al., Nature 313:810-812 (1985), the entirety of whichis herein incorporated by reference), the figwort mosaic virus35S-promoter, the light-inducible promoter from the small subunit ofribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh promoter(Walker et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:6624-6628 (1987), theentirety of which is herein incorporated by reference), the sucrosesynthase promoter (Yang et al., Proc. Natl. Acad. Sci. (U.S.A.)87:4144-4148 (1990), the entirety of which is herein incorporated byreference), the R gene complex promoter (Chandler et al., The Plant Cell1:1175-1183 (1989), the entirety of which is herein incorporated byreference), and the chlorophyll a/b binding protein gene promoter, etc.These promoters have been used to create DNA constructs which have beenexpressed in plants; see, e.g., PCT publication WO 84/02913, hereinincorporated by reference in its entirety.

Promoters which are known or are found to cause transcription of DNA inplant cells can be used in the present invention. Such promoters may beobtained from a variety of sources such as plants and plant viruses. Itis preferred that the particular promoter selected should be capable ofcausing sufficient expression to result in the production of aneffective amount of the cytokinin pathway enzyme to cause the desiredphenotype. In addition to promoters which are known to causetranscription of DNA in plant cells, other promoters may be identifiedfor use in the current invention by screening a plant cDNA library forgenes which are selectively or preferably expressed in the targettissues or cells.

For the purpose of expression in source tissues of the plant, such asthe leaf, seed, root or stem, it is preferred that the promotersutilized in the present invention have relatively high expression inthese specific tissues. For this purpose, one may choose from a numberof promoters for genes with tissue- or cell-specific or -enhancedexpression. Examples of such promoters reported in the literatureinclude the chloroplast glutamine synthetase GS2 promoter from pea(Edwards et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:3459-3463 (1990),herein incorporated by reference in its entirety), the chloroplastfructose-1,6-biphosphatase (FBPase) promoter from wheat (Lloyd et al.,Mol. Gen. Genet. 225:209-216 (1991), herein incorporated by reference inits entirety), the nuclear photosynthetic ST-LS1 promoter from potato(Stockhaus et al., EMBO J. 8:2445-2451 (1989), herein incorporated byreference in its entirety), the phenylalanine ammonia-lyase (PAL)promoter and the chalcone synthase (CHS) promoter from Arabidopsisthaliana. Also reported to be active in photosynthetically activetissues are the ribulose-1,5-bisphosphate carboxylase (RbcS) promoterfrom eastern larch (Larix laricina), the promoter for the cab gene,cab6, from pine (Yamamoto et al., Plant Cell Physiol. 35:773-778 (1994),herein incorporated by reference in its entirety), the promoter for theCab-1 gene from wheat (Fejes et al., Plant Mol. Biol. 15:921-932 (1990),herein incorporated by reference in its entirety), the promoter for theCAB-1 gene from spinach (Lubberstedt et al., Plant Physiol. 104:997-1006(1994), herein incorporated by reference in its entirety), the promoterfor the cab 1R gene from rice (Luan et al., Plant Cell. 4:971-981(1992), the entirety of which is herein incorporated by reference), thepyruvate, orthophosphate dikinase (PPDK) promoter from Zea mays(Matsuoka et al., Proc. Natl. Acad. Sci. (U.S.A.) 90: 9586-9590 (1993),herein incorporated by reference in its entirety), the promoter for thetobacco Lhcb1*2 gene (Cerdan et al., Plant Mol. Biol. 33: 245-255.(1997), herein incorporated by reference in its entirety), theArabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit etal., Planta. 196: 564-570 (1995), herein incorporated by reference inits entirety), and the promoter for the thylacoid membrane proteins fromspinach (psad, psaF, psae, PC, FNR, atpC, atpD, cab, rbcS). Otherpromoters for the chlorophyl a/b-binding proteins may also be utilizedin the present invention, such as the promoters for LhcB gene and PsbPgene from white mustard (Sinapis alba; Kretsch et al., Plant Mol. Biol.28: 219-229 (1995), the entirety of which is herein incorporated byreference).

For the purpose of expression in sink tissues of the plant, such as thetuber of the potato plant, the fruit of tomato, or the seed of Zea mays,wheat, rice, and barley, it is preferred that the promoters utilized inthe present invention have relatively high expression in these specifictissues. A number of promoters for genes with tuber-specific or-enhanced expression are known, including the class I patatin promoter(Bevan et al., EMBO J. 8: 1899-1906 (1986); Jefferson et al., Plant Mol.Biol. 14: 995-1006 (1990), both of which are herein incorporated byreference in its entirety), the promoter for the potato tuber ADPGPPgenes, both the large and small subunits, the sucrose synthase promoter(Salanoubat and Belliard, Gene. 60: 47-56 (1987), Salanoubat andBelliard, Gene. 84: 181-185 (1989), both of which are incorporated byreference in their entirety), the promoter for the major tuber proteinsincluding the 22 kd protein complexes and proteinase inhibitors(Hannapel, Plant Physiol. 101: 703-704 (1993), herein incorporated byreference in its entirety), the promoter for the granule bound starchsynthase gene (GBSS) (Visser et al., Plant Mol. Biol. 17: 691-699(1991), herein incorporated by reference in its entirety), and otherclass I and II patatins promoters (Koster-Topfer et al., Mol Gen Genet.219: 390-396 (1989); Mignery et al., Gene. 62: 2744 (1988), both ofwhich are herein incorporated by reference in their entirety).

Other promoters can also be used to express a fructose 1,6 bisphosphatealdolase gene in specific tissues, such as seeds or fruits. The promoterfor β-conglycinin (Chen et al., Dev. Genet. 10: 112-122 (1989), hereinincorporated by reference in its entirety) or other seed-specificpromoters such as the napin and phaseolin promoters, can be used. Thezeins are a group of storage proteins found in Zea mays endosperm.Genomic clones for zein genes have been isolated (Pedersen et al., Cell29: 1015-1026 (1982), herein incorporated by reference in its entirety),and the promoters from these clones, including the 15 kD, 16 kD, 19 kD,22 kD, 27 kD, and gamma genes, could also be used. Other promoters knownto function, for example, in Zea mays, include the promoters for thefollowing genes: waxy, Brittle, Shrunken 2, Branching enzymes I and II,starch synthases, debranching enzymes, oleosins, glutelins, and sucrosesynthases. A particularly preferred promoter for Zea mays endospermexpression is the promoter for the glutelin gene from rice, moreparticularly the Osgt-1 promoter (Zheng et al., Mol. Cell. Biol. 13:5829-5842 (1993), herein incorporated by reference in its entirety).Examples of promoters suitable for expression in wheat include thosepromoters for the ADPglucose pyrophosphorylase (ADPGPP) subunits, thegranule bound and other starch synthases, the branching and debranchingenzymes, the embryogenesis-abundant proteins, the gliadins, and theglutenins. Examples of such promoters in rice include those promotersfor the ADPGPP subunits, the granule bound and other starch synthases,the branching enzymes, the debranching enzymes, sucrose synthases, andthe glutelins. A particularly preferred promoter is the promoter forrice glutelin, Osgt-1. Examples of such promoters for barley includethose for the ADPGPP subunits, the granule bound and other starchsynthases, the branching enzymes, the debranching enzymes, sucrosesynthases, the hordeins, the embryo globulins, and the aleurone specificproteins.

Root specific promoters may also be used. An example of such a promoteris the promoter for the acid chitinase gene (Samac et al., Plant Mol.Biol. 25: 587-596 (1994), the entirety of which is herein incorporatedby reference). Expression in root tissue could also be accomplished byutilizing the root specific subdomains of the CaMV35S promoter that havebeen identified (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.)86:7890-7894 (1989), herein incorporated by reference in its entirety).Other root cell specific promoters include those reported by Conkling etal. (Conkling et al., Plant Physiol. 93:1203-1211 (1990), the entiretyof which is herein incorporated by reference).

Additional promoters that may be utilized are described, for example, inU.S. Pat. Nos. 5,378,619, 5,391,725, 5,428,147, 5,447,858, 5,608,144,5,608,144, 5,614,399, 5,633,441, 5,633,435, and 4,633,436, all of whichare herein incorporated in their entirety. In addition, a tissuespecific enhancer may be used (Fromm et al., The Plant Cell 1:977-984(1989), the entirety of which is herein incorporated by reference).

Constructs or vectors may also include, with the coding region ofinterest, a nucleic acid sequence that acts, in whole or in part, toterminate transcription of that region. For example, such sequences havebeen isolated including the Tr7 3′ sequence and the nos 3′ sequence(Ingelbrecht et al., The Plant Cell 1:671-680 (1989), the entirety ofwhich is herein incorporated by reference; Bevan et al., Nucleic AcidsRes. 11:369-385 (1983), the entirety of which is herein incorporated byreference), or the like.

A vector or construct may also include regulatory elements. Examples ofsuch include the Adh intron 1 (Callis et al., Genes and Develop.1:1183-1200 (1987), the entirety of which is herein incorporated byreference), the sucrose synthase intron (Vasil et al., Plant Physiol.91:1575-1579 (1989), the entirety of which is herein incorporated byreference) and the TMV omega element (Gallie et al., The Plant Cell1:301-311 (1989), the entirety of which is herein incorporated byreference). These and other regulatory elements may be included whenappropriate.

A vector or construct may also include a selectable marker. Selectablemarkers may also be used to select for plants or plant cells thatcontain the exogenous genetic material. Examples of such include, butare not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet.199:183-188 (1985), the entirety of which is herein incorporated byreference) which codes for kanamycin resistance and can be selected forusing kanamycin, G418, etc.; a bar gene which codes for bialaphosresistance; a mutant EPSP synthase gene (Hinchee et al., Bio/Technology6:915-922 (1988), the entirety of which is herein incorporated byreference) which encodes glyphosate resistance; a nitrilase gene whichconfers resistance to bromoxynil (Stalker et al., J. Biol. Chem.263:6310-6314 (1988), the entirety of which is herein incorporated byreference); a mutant acetolactate synthase gene (ALS) which confersimidazolinone or sulphonylurea resistance (European Patent Application154,204 (Sep. 11, 1985), the entirety of which is herein incorporated byreference); and a methotrexate resistant DHFR gene (Thillet et al., J.Biol. Chem. 263:12500-12508 (1988), the entirety of which is hereinincorporated by reference).

A vector or construct may also include a transit peptide. Incorporationof a suitable chloroplast transit peptide may also be employed (EuropeanPatent Application Publication Number 0218571, the entirety of which isherein incorporated by reference). Translational enhancers may also beincorporated as part of the vector DNA. DNA constructs could contain oneor more 5′ non-translated leader sequences which may serve to enhanceexpression of the gene products from the resulting mRNA transcripts.Such sequences may be derived from the promoter selected to express thegene or can be specifically modified to increase translation of themRNA. Such regions may also be obtained from viral RNAs, from suitableeukaryotic genes, or from a synthetic gene sequence. For a review ofoptimizing expression of transgenes, see Koziel et al., Plant Mol. Biol.32:393-405 (1996), the entirety of which is herein incorporated byreference.

A vector or construct may also include a screenable marker. Screenablemarkers may be used to monitor expression. Exemplary screenable markersinclude a β-glucuronidase or uidA gene (GUS) which encodes an enzyme forwhich various chromogenic substrates are known (Jefferson, Plant Mol.Biol, Rep. 5: 387-405 (1987), the entirety of which is hereinincorporated by reference; Jefferson et al., EMBO J. 6: 3901-3907(1987), the entirety of which is herein incorporated by reference); anR-locus gene, which encodes a product that regulates the production ofanthocyanin pigments (red color) in plant tissues ((Dellaporta et al.,Stadler Symposium 11:263-282 (1988), the entirety of which is hereinincorporated by reference); a β-lactamase gene (Sutcliffe et al., Proc.Natl. Acad. Sci. (U.S.A.) 75: 3737-3741 (1978), the entirety of which isherein incorporated by reference), a gene which encodes an enzyme forwhich various chromogenic substrates are known (e.g., PADAC, achromogenic cephalosporin); a luciferase gene (Ow et al., Science 234:856-859 (1986), the entirety of which is herein incorporated byreference) a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A.)80:1101-1105 (1983), the entirety of which is herein incorporated byreference) which encodes a catechol diozygenase that can convertchromogenic catechols; an α-amylase gene (Ikatu et al., Bio/Technol.8:241-242 (1990), the entirety of which is herein incorporated byreference); a tyrosinase gene (Katz et al., J. Gen. Microbiol.129:2703-2714 (1983), the entirety of which is herein incorporated byreference) which encodes an enzyme capable of oxidizing tyrosine to DOPAand dopaquinone which in turn condenses to melanin; an α-galactosidase,which will turn a chromogenic α-galactose substrate.

Included within the terms “selectable or screenable marker genes” arealso genes which encode a scriptable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes which canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA, small active enzymes detectable in extracellular solution (e.g.,α-amylase, β-lactamase, phosphinothricin transferase), or proteins whichare inserted or trapped in the cell wall (such as proteins which includea leader sequence such as that found in the expression unit of extensionor tobacco PR-S). Other possible selectable and/or screenable markergenes will be apparent to those of skill in the art.

Methods and compositions for transforming a bacteria and othermicroorganisms are known in the art (see for example Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989), the entiretyof which is herein incorporated by reference).

There are many methods for introducing transforming nucleic acidmolecules into plant cells. Suitable methods are believed to includevirtually any method by which nucleic acid molecules may be introducedinto a cell, such as by Agrobacterium infection or direct delivery ofnucleic acid molecules such as, for example, by PEG-mediatedtransformation, by electroporation or by acceleration of DNA coatedparticles, etc. (Pottykus, Ann. Rev. Plant Physiol. Plant Mol. Biol.42:205-225 (1991), the entirety of which is herein incorporated byreference; Vasil, Plant Mol. Biol. 25: 925-937 (1994), the entirety ofwhich is herein incorporated by reference. For example, electroporationhas been used to transform Zea mays protoplasts (Fromm et al., Nature312:791-793 (1986), the entirety of which is herein incorporated byreference).

Other vector systems suitable for introducing transforming DNA into ahost plant cell includes but is not limited to binary artificialchromosome (BIBAC) vectors (Hamilton et al., Gene 200:107-116, (1997),the entirety of which is herein incorporated by reference, andtransfection with RNA viral vectors (Della-Cioppa et al., Ann. N.Y.Acad. Sci. (1996), 792 (Engineering Plants for Commercial Products andApplications), 57-61, the entirety of which is herein incorporated byreference.

Technology for introduction of DNA into cells is well known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb,Virology, 54:536-539 (1973), the entirety of which is hereinincorporated by reference); (2) physical methods such as microinjection(Capecchi, Cell 22:479-488 (1980), electroporation (Wong and Neumann,Biochem. Biophys. Res. Commun., 107:584-587 (1982); Fromm et al., Proc.Natl. Acad. Sci. USA, 82:5824-5828 (1985); U.S. Pat. No. 5,384,253; andthe gene gun (Johnston and Tang, Methods Cell Biol. 43:353-365 (1994),all of which the entirety is herein incorporated by reference; (3) viralvectors (Clapp, Clin. Perinatol., 20:155-168 (1993); Lu et al., J. Exp.Med., 178:2089-2096 (1993); Eglitis and Anderson, Biotechniques,6:608-614 (1988), all of which the entirety is herein incorporated byreference); and (4) receptor-mediated mechanisms (Curiel et al., Hum.Gen. Ther., 3:147-154 (1992); Wagner et al., Proc. Natl. Acad. Sci. USA,89:6099-6103 (1992), all of which the entirety is herein incorporated byreference).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang andChristou, eds., Particle Bombardment Technology for Gene Transfer,Oxford Press, Oxford, England (1994), the entirety of which is hereinincorporated by reference). Non-biological particles (microprojectiles)that may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

A particular advantage of microprojectile bombardment, in addition to itbeing an effective means of reproducibly, and stably transformingmonocotyledons, is that neither the isolation of protoplasts (Cristou etal., Plant Physiol. 87:671-674 (1988), the entirety of which is hereinincorporated by reference) nor the susceptibility of Agrobacteriuminfection is required. An illustrative embodiment of a method fordelivering DNA into maize cells by acceleration is a biolisticsγ-particle delivery system, which can be used to propel particles coatedwith DNA through a screen, such as a stainless steel or Nytex screen,onto a filter surface covered with corn cells cultured in suspension.Gordon-Kamm et al., describes the basic procedure for coating tungstenparticles with DNA (Gordon-Kamm et al., Plant Cell 2: 603-618 (1990),the entirety of which is herein incorporated by reference). The screendisperses the tungsten nucleic acid particles so that they are notdelivered to the recipient cells in large aggregates. A particledelivery system suitable for use with the present invention is thehelium acceleration PDS-1000/He gun which is available from Bio-RadLaboratories (Bio-Rad, Hercules, Calif.) (Sanford et al., Technique3:3-16 (1991), the entirety of which is herein incorporated byreference).

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the macroprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain up to 1000 or more foci ofcells transiently expressing a marker gene. The number of cells in afocus which express the exogenous gene product 48 hours post-bombardmentoften range from one to ten and average one to three.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

In another alternative embodiment, plastids can be stably transformed.Methods disclosed for plastid transformation in higher plants includethe particle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (Svab et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8526-8530(1990); Svab and Maliga, Proc. Natl. Acad. Sci. (U.S.A.) 90:913-917(1993); Staub and Maliga, EMBO J. 12:601-606 (1993); U.S. Pat. Nos.5,451,513 and 5,545,818, all of which are herein incorporated byreference in their entirety).

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions which influence the physiological state of therecipient cells and which may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example the methods described (Fraley et al.,Biotechnology 3:629-635 (1985); Rogers et al., Meth. In Enzymol,153:253-277 (1987), both of which are herein incorporated by referencein their entirety. Further, the integration of the Ti-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., Mol. Gen. Genet., 205:34 (1986), the entirety ofwhich is herein incorporated by reference).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, T. Hohn and J. Schell, eds., Springer-Verlag, New York, pp.179-203 (1985), the entirety of which is herein incorporated byreference. Moreover, recent technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes (Rogerset al., Meth. In Enzymol., 153:253-277 (1987), the entirety of which isherein incorporated by reference). In addition, Agrobacterium containingboth armed and disarmed Ti genes can be used for the transformations. Inthose plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene. Morepreferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes thatencode a polypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. See for example(Potrykus et al., Mol. Gen. Genet., 205:193-200 (1986); Lorz et al.,Mol. Gen. Genet., 199:178, (1985); Fromm et al., Nature, 319:791,(1986); Uchimiya et al., Mol. Gen. Genet.: 204:204, (1986); Callis etal., Genes and Development, 1183, (1987); Marcotte et al., Nature,335:454, (1988), all of which the entirety is herein incorporated byreference).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., Plant Tissue Culture Letters, 2:74,(1985); Toriyama et al., Theor Appl. Genet. 205:34. (1986); Yamada etal., Plant Cell Rep., 4:85, (1986); Abdullah et al., Biotechnology,4:1087, (1986), all of which the entirety is herein incorporated byreference).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, Biotechnology,6:397, (1988), the entirety of which is herein incorporated byreference). In addition, “particle gun” or high-velocity microprojectiletechnology can be utilized (Vasil et al., Bio/technology 10:667, (1992),the entirety of which is herein incorporated by reference).

Using the latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., Nature, 328:70, (1987); Klein et al., Proc. Natl. Acad.Sci. USA, 85:8502-8505, (1988); McCabe et al., Biotechnology, 6:923,(1988), all of which the entirety is herein incorporated by reference).The metal particles penetrate through several layers of cells and thusallow the transformation of cells within tissue explants.

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen (Zhou et al., Methods in Enzymology, 101:433,(1983); Hess et al., Intern Rev. Cytol., 107:367, (1987); Luo et al.,Plant Mol. Biol. Reporter, 6:165, (1988), all of which the entirety isherein incorporated by reference), by direct injection of DNA intoreproductive organs of a plant (Pena et al., Nature, 325:274, (1987),the entirety of which is herein incorporated by reference), or by directinjection of DNA into the cells of immature embryos followed by therehydration of dessicated embryos (Neuhaus et al., Theor. Appl. Genet.,75:30, (1987), the entirety of which is herein incorporated byreference).

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, In: Methods for PlantMolecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif.,(1988), the entirety of which is herein incorporated by reference). Thisregeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important lines. Conversely, pollen from plants of theseimportant lines is used to pollinate regenerated plants. A transgenicplant of the present invention containing a desired polypeptide iscultivated using methods well known to one skilled in the art.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No.5,518,908, all of which the entirety is herein incorporated byreference); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011,McCabe et al., Biotechnology 6:923, (1988), Christou et al., PlantPhysiol., 87:671-674 (1988), all of which the entirety is hereinincorporated by reference); Brassica (U.S. Pat. No. 5,463,174, theentirety of which is herein incorporated by reference); peanut (Cheng etal., Plant Cell Rep. 15: 653-657 (1996), McKently et al., Plant CellRep. 14:699-703 (1995), all of which the entirety is herein incorporatedby reference); papaya (Yang et al., (1996), the entirety of which isherein incorporated by reference); pea (Grant et al., Plant Cell Rep.15:254-258, (1995), the entirety of which is herein incorporated byreference).

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. USA 84:5345, (1987), the entirety of which isherein incorporated by reference); barley (Wan and Lemaux, Plant Physiol104:37, (1994), the entirety of which is herein incorporated byreference); maize (Rhodes et al., Science 240: 204, (1988), Gordon-Kammet al., Plant Cell, 2:603, (1990), Fromm et al., Bio/Technology 8:833,(1990), Koziel et al., Bio/Technology 11:194, (1993), Armstrong et al.,Crop Science 35:550-557, (1995), all of which the entirety is hereinincorporated by reference); oat (Somers et al., Bio/Technology, 10:1589,(1992), the entirety of which is herein incorporated by reference);orchardgrass (Horn et al., Plant Cell Rep. 7:469, (1988), the entiretyof which is herein incorporated by reference); rice (Toriyama et al.,Theor Appl. Genet. 205:34, (1986); Park et al., Plant Mol. Biol., 32:1135-1148, (1996); Abedinia et al., Aust. J. Plant Physiol. 24: 133-141,(1997); Zhang and Wu, Theor. Appl. Genet. 76:835, (1988); Zhang et al.Plant Cell Rep. 7:379, (1988); Battraw and Hall, Plant Sci. 86:191-202,(1992); Christou et al., Bio/Technology 9:957, (1991), all of which theentirety is herein incorporated by reference); sugarcane (Bower andBirch, Plant J. 2:409, (1992), the entirety of which is hereinincorporated by reference); tall fescue (Wang et al., Bio/Technology10:691, (1992), the entirety of which is herein incorporated byreference), and wheat (Vasil et al., Bio/Technology 10:667, (1992), theentirety of which is herein incorporated by reference; U.S. Pat. No.5,631,152, the entirety of which is herein incorporated by reference.

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte, et al., Nature, 335:454-457 (1988), the entirety of which is herein incorporated byreference; Marcotte, et al., Plant Cell, 1: 523-532 (1989), the entiretyof which is herein incorporated by reference; McCarty, et al., Cell 66:895-905 (1991), the entirety of which is herein incorporated byreference; Hattori, et al., Genes Dev. 6: 609-618 (1992), the entiretyof which is herein incorporated by reference; Goff, et al., EMBO J. 9:2517-2522 (1990), the entirety of which is herein incorporated byreference). Transient expression systems may be used to functionallydissect gene constructs (See generally, Mailga et al., Methods in PlantMolecular Biology, Cold Spring Harbor Press (1995)).

Any of the nucleic acid molecules of the present invention may beintroduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promotersenhancers etc. Further any of the nucleic acid molecules of the presentinvention may be introduced into a plant cell in a manner that allowsfor over expression of the protein or fragment thereof encoded by thenucleic acid molecule.

Cosuppression is the reduction in expression levels, usually at thelevel of RNA, of a particular endogenous gene or gene family by theexpression of a homologous sense construct that is capable oftranscribing mRNA of the same strandedness as the transcript of theendogenous gene (Napoli et al., Plant Cell 2: 279-289 (1990), theentirety of which is herein incorporated by reference; van der Krol etal., Plant Cell 2: 291-299 (1990), the entirety of which is hereinincorporated by reference). Cosuppression may result from stabletransformation with a single copy nucleic acid molecule that ishomologous to a nucleic acid sequence found with the cell (Prolls andMeyer, Plant J. 2:465-475 (1992), the entirety of which is hereinincorporated by reference) or with multiple copies of a nucleic acidmolecule that is homologous to a nucleic acid sequence found with thecell (Mittlesten et al., Mol. Gen. Genet. 244: 325-330 (1994), theentirety of which is herein incorporated by reference). Genes, eventhough different, linked to homologous promoters may result in thecosuppression of the linked genes (Vaucheret, C.R. Acad. Sci. III 316:1471-1483 (1993), the entirety of which is herein incorporated byreference).

This technique has, for example been applied to generate white flowersfrom red petunia and tomatoes that do not ripen on the vine. Up to 50%of petunia transformants that contained a sense copy of the chalconesynthase (CHS) gene produced white flowers or floral sectors; this wasas a result of the post-transcriptional loss of mRNA encoding CHS(Flavell, Proc. Natl. Acad. Sci. (U.S.A.) 91:3490-3496 (1994)), theentirety of which is herein incorporated by reference). Cosuppressionmay require the coordinate transcription of the transgene and theendogenous gene, and can be reset by a developmental control mechanism(Jorgensen, Trends Biotechnol, 8:340344 (1990), the entirety of which isherein incorporated by reference; Meins and Kunz, In: Gene Inactivationand Homologous Recombination in Plants (Paszkowski, J., ed.), pp.335-348. Kluwer Academic, Netherlands (1994), the entirety of which isherein incorporated by reference).

It is understood that one or more of the nucleic acids of the presentinvention comprising SEQ ID NO:1 or complement thereof through SEQ IDNO:5521 or complement thereof, may be introduced into a plant cell andtranscribed using an appropriate promoter with such transcriptionresulting in the co-suppression of an endogenous protein.

Antisense approaches are a way of preventing or reducing gene functionby targeting the genetic material (Mol et al., FEBS Lett. 268: 427-430(1990), the entirety of which is herein incorporated by reference). Theobjective of the antisense approach is to use a sequence complementaryto the target gene to block its expression and create a mutant cell lineor organism in which the level of a single chosen protein is selectivelyreduced or abolished. Antisense techniques have several advantages overother ‘reverse genetic’ approaches. The site of inactivation and itsdevelopmental effect can be manipulated by the choice of promoter forantisense genes or by the timing of external application ormicroinjection. Antisense can manipulate its specificity by selectingeither unique regions of the target gene or regions where it shareshomology to other related genes (Hiatt et al., In Genetic Engineering,Setlow (ed.), Vol. 11, New York: Plenum 49-63 (1989), the entirety ofwhich is herein incorporated by reference).

The principle of regulation by antisense RNA is that RNA that iscomplementary to the target mRNA is introduced into cells, resulting inspecific RNA:RNA duplexes being formed by base pairing between theantisense substrate and the target mRNA (Green et al., Annu. Rev.Biochem. 55: 569-597 (1986), the entirety of which is hereinincorporated by reference). Under one embodiment, the process involvesthe introduction and expression of an antisense gene sequence. Such asequence is one in which part or all of the normal gene sequences areplaced under a promoter in inverted orientation so that the ‘wrong’ orcomplementary strand is transcribed into a noncoding antisense RNA thathybridizes with the target mRNA and interferes with its expression(Takayama and Inouye, Crit. Rev. Biochem. Mol. Biol. 25: 155-184 (1990),the entirety of which is herein incorporated by reference). An antisensevector is constructed by standard procedures and introduced into cellsby transformation, transfection, electroporation, microinjection, or byinfection, etc. The type of transformation and choice of vector willdetermine whether expression is transient or stable. The promoter usedfor the antisense gene may influence the level, timing, tissue,specificity, or inducibility of the antisense inhibition.

It is understood that protein synthesis activity in a plant cell may bereduced or depressed by growing a transformed plant cell containing anucleic acid molecule whose non-transcribed strand encodes a proteinsynthesis enzyme or fragment thereof.

Antibodies have been expressed in plants (Hiatt et al., Nature 342:76-78(1989), the entirety of which is herein incorporated by reference;Conrad and Fielder, Plant Mol. Biol. 26: 1023-1030 (1994), the entiretyof which is herein incorporated by reference). Cytoplamic expression ofa scFv (single-chain Fv antibodies) has been reported to delay infectionby artichoke mottled crinkle virus. Transgenic plants that expressantibodies directed against endogenous proteins may exhibit aphysiological effect (Philips et al., EMBO J. 16: 4489-4496 (1997), theentirety of which is herein incorporated by reference; Marion-Poll,Trends in Plant Science 2: 447-448 (1997), the entirety of which isherein incorporated by reference). For example, expressed anti-abscisicantibodies reportedly result in a general perturbation of seeddevelopment (Philips et al., EMBO J. 16: 4489-4496 (1997)).

Antibodies that are catalytic may also be expressed in plants (abzymes).The principle behind abzymes is that since antibodies may be raisedagainst many molecules, this recognition ability can be directed towardgenerating antibodies that bind transition states to force a chemicalreaction forward (Persidas, Nature Biotechnology 15:1313-1315 (1997),the entirety of which is herein incorporated by reference; Baca et al.,Ann. Rev. Biophys. Biomol. Struct. 26:461-493 (1997), the entirety ofwhich is herein incorporated by reference). The catalytic abilities ofabzymes may be enhanced by site directed mutagensis. Examples of abzymesare, for example, set forth in U.S. Pat. No. 5,658,753; U.S. Pat. No.5,632,990; U.S. Pat. No. 5,631,137; U.S. Pat. No. 5,602,015; U.S. Pat.No. 5,559,538; U.S. Pat. No. 5,576,174; U.S. Pat. No. 5,500,358; U.S.Pat. No. 5,318,897; U.S. Pat. No. 5,298,409; U.S. Pat. No. 5,258,289 andU.S. Pat. No. 5,194,585, all of which are herein incorporated in theirentirety.

It is understood that any of the antibodies of the present invention maybe expressed in plants and that such expression can result in aphysiological effect. It is also understood that any of the expressedantibodies may be catalytic.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Mailga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995), theentirety of which is herein incorporated by reference; Birren et al.,Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y., theentirety of which is herein incorporated by reference).

The nucleotide sequence provided in SEQ ID NO: 1, through SEQ ID NO:5521or fragment thereof, or complement thereof, or a nucleotide sequence atleast 90% identical, preferably 95%, identical even more preferably 99%or 100% identical to the sequence provided in SEQ ID NO:1 through SEQ IDNO:5521 or fragment thereof, or complement thereof, can be “provided” ina variety of mediums to facilitate use fragment thereof. Such a mediumcan also provide a subset thereof in a form that allows a skilledartisan to examine the sequences.

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium that can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard disc,storage medium, and magnetic tape: optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable medium to generate media comprising the nucleotide sequenceinformation of the present invention. A variety of data storagestructures are available to a skilled artisan for creating a computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable medium. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and Microsoft Word,or represented in the form of an ASCII file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. A skilled artisancan readily adapt any number of data processor structuring formats (e.g.text file or database) in order to obtain computer readable mediumhaving recorded thereon the nucleotide sequence information of thepresent invention.

By providing one or more of nucleotide sequences of the presentinvention, a skilled artisan can routinely access the sequenceinformation for a variety of purposes. Computer software is publiclyavailable which allows a skilled artisan to access sequence informationprovided in a computer readable medium. The examples which followdemonstrate how software which implements the BLAST (Altschul et al., J.Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem.17:203-207 (1993), the entirety of which is herein incorporated byreference) search algorithms on a Sybase system can be used to identifyopen reading frames (ORFs) within the genome that contain homology toORFs or proteins from other organisms. Such ORFs are protein-encodingfragments within the sequences of the present invention and are usefulin producing commercially important proteins such as enzymes used inamino acid biosynthesis, metabolism, transcription, translation, RNAprocessing, nucleic acid and a protein degradation, proteinmodification, and DNA replication, restriction, modification,recombination, and repair.

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence information describedherein. Such systems are designed to identify commercially importantfragments of the nucleic acid molecule of the present invention. As usedherein, “a computer-based system” refers to the hardware means, softwaremeans, and data storage means used to analyze the nucleotide sequenceinformation of the present invention. The minimum hardware means of thecomputer-based systems of the present invention comprises a centralprocessing unit (CPU), input means, output means, and data storagemeans. A skilled artisan can readily appreciate that any one of thecurrently available computer-based system are suitable for use in thepresent invention.

As indicated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein a nucleotidesequence of the present invention and the necessary hardware means andsoftware means for supporting and implementing a search means. As usedherein, “data storage means” refers to memory that can store nucleotidesequence information of the present invention, or a memory access meanswhich can access manufactures having recorded thereon the nucleotidesequence information of the present invention. As used herein, “searchmeans” refers to one or more programs which are implemented on thecomputer-based system to compare a target sequence or target structuralmotif with the sequence information stored within the data storagemeans. Search means are used to identify fragments or regions of thesequence of the present invention that match a particular targetsequence or target motif. A variety of known algorithms are disclosedpublicly and a variety of commercially available software for conductingsearch means are available can be used in the computer-based systems ofthe present invention. Examples of such software include, but are notlimited to, MacPattern (EMBL), BLASTIN and BLASTIX (NCBIA). One of theavailable algorithms or implementing software packages for conductinghomology searches can be adapted for use in the present computer-basedsystems.

The most preferred sequence length of a target sequence is from about 10to 100 amino acids or from about 30 to 300 nucleotide residues. However,it is well recognized that during searches for commercially importantfragments of the nucleic acid molecules of the present invention, suchas sequence fragments involved in gene expression and proteinprocessing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequences the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein targetmotifs include, but are not limited to, enzymatic active sites andsignal sequences. Nucleic acid target motifs include, but are notlimited to, promoter sequences, cis elements, hairpin structures andinducible expression elements (protein binding sequences).

Thus, the present invention further provides an input means forreceiving a target sequence, a data storage means for storing the targetsequences of the present invention sequence identified using a searchmeans as described above, and an output means for outputting theidentified homologous sequences. A variety of structural formats for theinput and output means can be used to input and output information inthe computer-based systems of the present invention. A preferred formatfor an output means ranks fragments of the sequence of the presentinvention by varying degrees of homology to the target sequence ortarget motif. Such presentation provides a skilled artisan with aranking of sequences which contain various amounts of the targetsequence or target motif and identifies the degree of homology containedin the identified fragment.

A variety of comparing means can be used to compare a target sequence ortarget motif with the data storage means to identify sequence fragmentssequence of the present invention. For example, implementing softwarewhich implement the BLAST and BLAZE algorithms (Altschul et al., J. Mol.Biol. 215:403-410 (1990)) can be used to identify open frames within thenucleic acid molecules of the present invention. A skilled artisan canreadily recognize that any one of the publicly available homology searchprograms can be used as the search means for the computer-based systemsof the present invention. Having now generally described the invention,the same will be more readily understood through reference to thefollowing examples which are provided by way of illustration, and arenot intended to be limiting of the present invention, unless specified.

EXAMPLE 1

The cDNA library of the present invention designated SOYMON001, wasprepared from soybean cultivar Asgrow 3244. This cDNA library wasgenerated from leaves from V4 stage plants. The term V4 as used hereinrefers to the vegetative stage of plant development for soybean in whichthe plant contains four fully developed leaf nodes. The vegetativestages of soybean development are subdivided and designated V1, V2, V3,through V(n), except for the first two stages which are designated as VE(emergence) and VC (cotyledon stage). The last V stage is designated asV(n), where (n) represents the number for the last node stage of thespecific variety. The (n) fluctuates with variety and environmentalconditions. These designations are well known to those of skill in theart (see, for example, How a Soybean Plant Develops, (1996), specialreport No. 53, Iowa State University of Science and Technology,Cooperative Extension Service, Ames, Iowa)

The plants were grown under field conditions and the leaf tissue washarvested from the 4^(th) node. The tissue was removed from the plantsin the field, placed in 15 ml culture tubes and immersed in dry-ice. Thetissue was subsequently transferred to a −80° C. freezer and storeduntil the RNA isolation was initiated.

For the cDNA library of the present invention, the Superscript™ PlasmidSystem for cDNA synthesis and Plasmid Cloning (Gibco BRL, LifeTechnologies, Gaithersburg, Md.) or similar system is used, followingthe conditions suggested by the manufacturer. PolyA mRNA is purifiedfrom the total RNA preparation using Dynabeads® Oligo(dT)₂₅ (Dynal Inc.,Lake Success, N.Y.), or equivalent methods.

The quality of the cDNA libraries is determined by examining the cDNAinsert size, and also by sequence analysis of a random selection anappropriate number of clones from the library.

EXAMPLE 2

The cDNA library of the present invention, SOYMON001, is plated on LBagar containing the appropriate antibiotics for selection and incubatedat 37° C. for a sufficient time to allow the growth of individualcolonies. Single colonies are individually placed in each well of96-well microtiter plates containing LB liquid including the selectiveantibiotics. The plates are incubated overnight at approximately 37° C.with gentle shaking to promote growth of the cultures. The plasmid DNAis isolated from each clone using a commercially available kit such asQiaprep plasmid isolation kits, using the conditions recommended by themanufacturer (Qiagen Inc., Santa Clarita, Calif.). A variety of plasmidisolation kits are commercially available.

The template plasmid DNA clones are used for subsequent sequencing. Forsequencing the cDNA library SOYMON001, a commercially availablesequencing kit, such as the ABI PRISM dRhodamine Terminator CycleSequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS, is usedunder the conditions recommended by the manufacturer (PE AppliedBiosystems, Foster City, Calif.). The ESTs of the present invention aregenerated by sequencing initiated from the 5′ end of each cDNA clone.

A number of sequencing techniques are known in the art, includingfluorescence-based sequencing methodologies. These methods have thedetection, automation and instrumentation capability necessary for theanalysis of large volumes of sequence data. Currently, the 377 DNASequencer (Perkin-Elmer Corp., Applied Biosystems Div., Foster City,Calif.) allows the most rapid electrophoresis and data collection. Withthese types of automated systems, fluorescent dye-labeled sequencereaction products are detected and data entered directly into thecomputer, producing a chromatogram that is subsequently viewed, stored,and analyzed using the corresponding software programs. These methodsare known to those of skill in the art and have been described andreviewed (Birren et al., Genome Analysis: Analyzing DNA, 1, Cold SpringHarbor, N.Y., the entirety of which is herein incorporated byreference).

1. A substantially purified nucleic acid molecule that encodes a soybeanprotein or fragment thereof comprising a sequence selected from thegroup consisting of SEQ ID NO: 1 through SEQ ID NO:
 5521. 2. Asubstantially purified soybean protein or fragment thereof, wherein saidsoybean protein is encoded by a nucleic acid molecule that comprises anucleic acid sequence selected from the group consisting of SEQ ID NO: 1through SEQ ID NO:
 5521. 3. A substantially purified antibody orfragment thereof which is capable of specifically binding to a specificmaize or soybean enzyme or fragment thereof according to claim
 2. 4. Atransformed plant having a nucleic acid molecule which comprises: (a) anexogenous promoter region which functions in a plant cell to cause theproduction of a mRNA molecule; (b) a structural nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1 through SEQ ID NO: 5521 or complements thereof; (c) a 3′non-translated sequence that functions in said plant cell to causetermination of transcription and addition of polyadenylatedribonucleotides to a 3′ end of said mRNA molecule.
 5. The transformedplant according to claim 4, wherein said structural nucleic acidmolecule is a complement of any of the nucleic acid sequences of SEQ IDNO: 1 through SEQ ID NO:
 5521. 6. The transformed plant according toclaim 5, wherein said plant is soybean or maize.
 7. The transformedplant according to claim 5, wherein said plant is soybean.
 8. A methodfor determining a level or pattern in a plant cell of a protein in aplant comprising: (a) incubating, under conditions permitting nucleicacid hybridization, a marker nucleic acid molecule, said marker nucleicacid molecule selected from the group of marker nucleic acid moleculeswhich specifically hybridize to a nucleic acid molecule having thenucleic acid sequence selected from the group consisting of SEQ ID NO: 1through SEQ ID NO: 5521 or complement thereof, with a complementarynucleic acid molecule obtained from said plant cell or plant tissue,wherein nucleic acid hybridization between said marker nucleic acidmolecule and said complementary nucleic acid molecule obtained from saidplant cell or plant tissue permits the detection of an mRNA for saidenzyme; (b) permitting hybridization between said marker nucleic acidmolecule and said complementary nucleic acid molecule obtained from saidplant cell or plant tissue; and (c) detecting the level or pattern ofsaid complementary nucleic acid, wherein the detection of saidcomplementary nucleic acid is predictive of the level or pattern of saidprotein.
 9. The method of claim 8, wherein said level or pattern isdetected by in situ hybridization.
 10. The method of claim 8, whereinsaid level or pattern is detected by tissue printing.
 11. The method ofclaim 8, wherein said plant is maize or soybean.
 12. The method of claim11, wherein said plant is soybean.
 13. A method of determining amutation in a plant whose presence is predictive of a mutation affectinga level or pattern of a protein comprising the steps: (a) incubating,under conditions permitting nucleic acid hybridization, a marker nucleicacid, said marker nucleic acid selected from the group of marker nucleicacid molecules which specifically hybridize to a nucleic acid moleculeconsisting of the nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1 through SEQ ID NO: 5521 or complement thereofand a complementary nucleic acid molecule obtained from said plant,wherein nucleic acid hybridization between said marker nucleic acidmolecule and said complementary nucleic acid molecule obtained from saidplant permits the detection of a polymorphism whose presence ispredictive of a mutation affecting said level or pattern of said proteinin said plant; (b) permitting hybridization between said marker nucleicacid molecule and said complementary nucleic acid molecule obtained fromsaid plant; and (c) detecting the presence of said polymorphism, whereinthe detection of said polymorphism is predictive of said mutation. 14.The method of claim 13, wherein said level or pattern is detected by insitu hybridization.
 15. The method of claim 13, wherein said level orpattern is detected by tissue printing.
 16. The method of claim 13,wherein said plant is maize or soybean.
 17. The method of claim 16,wherein said plant is soybean.